Compositions for Enhancing Segregation of Transgenes in Plants

ABSTRACT

The compositions and methods are provided that enhance the selection of transgenic plants having two T-DNA molecules integrated into a plant genome at different physical and genetic loci. The compositions are DNA constructs that comprise novel arrangements of T-DNA molecules containing genes of interest, positive selectable marker genes, and conditional lethal genes. The methods disclosed herein comprises transforming a plant cell to comprise the DNA constructs of the present invention, regenerating the plant cell into a plant and identifying independant transgene loci, where the selectable marker genes or transgenic elements can be segregated in the progeny.

FIELD OF THE INVENTION

The present invention relates to the field of plant genetic engineering.Particularly, the invention relates to DNA constructs and methods thatenhance the segregation of transgenes in plants.

BACKGROUND OF THE INVENTION

The methods for introducing transgenes in plants by Agrobacteriummediated transformation utilizes a T-DNA (transfer DNA) thatincorporates the genetic elements of the transgene and transfers thosegenetic elements into the genome of a plant. Generally, the transgene(s)bordered by a right border DNA molecule (RB) and a left border DNAmolecule (LB) is transferred into the plant genome at a single locus. Ithas been previously observed that when a DNA construct contains morethan one T-DNA these T-DNAs and the transgenes contained within may beintegrated into the plant genome at separate loci and is referred to aco-transformation (U.S. Pat. No. 5,731,179, WO 00/18939). The process ofco-transformation, where two T-DNAs are at different loci in the plantgenome and therefore segregate independently in the progeny, can beachieved by delivery of the T-DNAs with a mixture of Agrobacteriatransformed with plasmids carrying the separate T-DNA (Depicker et al.,Mol. Gen. Genet. 201:477-484, 1985; Petit et al., Mol. Gen. Genet.202:388-393, 1986; McKnight et al., Plant Mol. Biol. 8:439-445, 1987;DeBlock et al., Theor. Appl. Genet. 82:257-263, 1991; Komari et al.,Plant J. 10: 165-174, 1996; De Neve et al., Plant J. 11: 15-29, 1997;Poirier et al., Theor. Appl. Genet. 100:487-493, 2000, hereinincorporated by reference in their entirety). Co-transformation can alsobe achieved by transforming one Agrobacterium strain with two binary DNAconstructs, each containing one T-DNA (Daley et al., Plant Cell Reports17:489-496 1998). An additional method employs constructing the twoT-DNAs on a single DNA vector, transforming the vector into a plant celland then identifying the transgenic cells or plants that have integratedthe T-DNAs at different loci (U.S. Pat. No. 5,731,179, WO 00/18939,Komari et al., Plant J. 10: 165-174, 1996; Xing et al., In Vitro CellDevel. Biol. Plant 36:456-463, 2000).

Unlinking T-DNAs that contain different plant expression cassettes canbe useful in the development of transgenic plants, especially if one ofthe plant expression cassettes does not contribute to an agronomicallyuseful trait introduced into the crop plant. It has been previouslyobserved that Agrobacterium Ti plasmid T-DNAs can insert into the genomeof a transformed plant at more than one loci and will segregate in theprogeny of the plant (Framond et al., Mol. Gen. Genet. 202:125-131,1986). The identification of unnecessary or unwanted transgene DNA intransformed plants has been the subject of numerous investigations andmany different methods have been examined in efforts to eliminate thesetransgenes or vector DNA from the plants (Hanson et al., Plant J.19:727-734, 1999; Dale et al., Proc. Natl. Acad. Sci. 88:10558-10562,1991; Ebinuma et al., Proc. Natl. Acad. Sci. 94:2117-2121, 1997; Yoderet al., Bio/Technology 12:263-268, 1994; Kononov et al., Plant J.11:945-957, 1997).

A two T-DNA system is a useful method to segregate the marker gene fromthe agronomically important gene of interest (GOI) in a transgenicplant. The marker gene generally has no further utility after it hasbeen used to select or score for the transformed plant cell. Theresearch effort to develop an efficient two T-DNA system that providesseparate and distinct genomic integration sites has been extensive. Asingle DNA vector carrying the two-T-DNAs is the preferred method toconstruct a two T-DNA transformation system. However, because of theoccurrence of both T-DNAs on a single DNA construct often both aretransferred into the plant genome at the same loci. This occurs when oneof the border DNA molecule of the first T-DNA is not recognized duringthe integration process. This reduced efficiency adds to the cost ofproducing the events and selecting for the individuals that have T-DNAsintegrated at an independent locus. It would be of general utility tohave DNA constructs and a method where it is possible to chemicallyselect against individuals that have incorporated the two T-DNAs at thesame loci or have incorporated unnecessary DNA that occurs in the spacerregion between the two T-DNAs, or unnecessary DNA that occurs in theregion of the DNA construct flanking the two T-DNAs.

Heterologous dominant conditional lethal genes have the greatestpotential for controlled cell lethality. The genes are by definition,non-lethal in the absence of the controlled application of aheterologous protoxin and utilize protoxins which are not substrates fornormal cellular enzymes. In plants, some examples of heterologousconditional lethal genes are the pehA gene that converts a nontoxicphosphonate of glyphosate into glyphosate (U.S. Pat. No. 5,254,801, theentirety of which is herein incorporated by reference), argE gene thatconverts a nontoxic N-acetyl-phosphinothricin to toxic phosphinothricin(Kriete et al. Plant J. 9:809-818, 1996, the entirety of which is hereinincorporated by reference), the iaah gene encoding indoleacetamidehydrolase which can convert non-toxic levels of naphthalene acetamideinto toxic levels of the auxin, naphthalene acetic acid (Klee et al.,Genes Dev. 1:86-96, 1987). A bacterial cytosine deaminase gene has beenshown to function as a conditional lethal gene for negative selection inplants (Kobayashi et al. Jpn. J. Genet. 70:409-422; Perera et al. PlantMol. Biol. 23:793-799, 1993, the entirety of which is hereinincorporated by reference). P450 monooxygenase expression in plant cellsconverts a sulphonylurea compound (R7402) with low toxicity into a morehighly phytotoxic form (O'Keefe et al. Plant Physiol. 105:473-482, 1994,the entirety of which is herein incorporated by reference). Transgenicplants expressing β-glucuronidase in male tissues can convert aglucuronic acid conjugated protoxin into a phytotoxic molecule providinga gametocide (WO9204454, the entirety of which is herein incorporated byreference). Viral thymidine kinase is an example of a heterologousdominant conditional lethal gene in mammalian cells the functions inplants (Czako et al. Plant Physiol. 104:1067-1071, 1994). Unlike thecellular thymidine kinase, the viral thymidine kinase protein is able toactivate pyrimidine analogs such as acycolvir and gancyclovia into toxicproducts. The most effective conditional lethal gene enzymes have noaffect on cellular metabolism in the absence of the protoxin andutilizes a protoxin that is at least several fold less toxic than theactivated toxin.

The present invention provides a conditional lethal transgene in the twoT-DNA construct and a method to select against a plant cell, plant orprogeny thereof that express a conditional lethal phenotype. Aconditional lethal phenotype involves the expression of a gene productthat is not lethal to a cell under normal conditions. To this end theconditional lethal phenotype is used to eliminate the unwanted plantcells, plant tissues or plants when it is most desirable to do so. Thepresent invention provides DNA constructs in novel combinations withpositive selectable marker transgenes and methods for the use of theseDNA constructs with or without a conditional lethal phenotype in a twoT-DNA system for plants. The methods of the present invention enhancethe identification and selection of transgenic events in which the twoT-DNA segments have integrated into the plant genome at differentphysical and genetic loci, and provide transgenic plants withagronomically useful transgenes and phenotypes free of the markertransgenes.

SUMMARY OF THE INVENTION

The present invention provides a DNA construct comprising at least twoT-DNA molecules and at least two DNA segments separating said T-DNAmolecules, wherein a conditional lethal gene resides in at least one DNAsegment. In another aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein a conditional lethal generesides in each DNA segment.

In another aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein a first conditional lethal generesides in any one DNA segment and a second conditional lethal geneheterologous to said first conditional lethal gene resides in any one ofthe T-DNA molecules. In a further aspect of the invention, a DNAconstruct is provided comprising at least two T-DNA molecules and atleast two DNA segments separating said T-DNA molecules, wherein a firstconditional lethal gene resides in one DNA segment and a secondconditional lethal gene resides in the second DNA segment; and a thirdconditional lethal gene heterologous to said first and secondconditional lethal gene resides in any one of the T-DNA molecules. In afurther aspect of the invention, the expression of a conditional lethalgene residing in a T-DNA molecule is under the control of a tissuespecific or inducible promoter.

In a further aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein one of the T-DNA moleculescontains a selectable marker gene and a conditional lethal gene. Inanother aspect of the invention, a DNA construct is provided comprisingat least two T-DNA molecules and at least two DNA segments separatingsaid T-DNA molecules, wherein one of the T-DNA molecules contains aselectable marker gene and a first conditional lethal gene; and a secondconditional lethal gene heterologous to said first conditional lethalgene resides in any one DNA segment. In still another aspect of theinvention, a DNA construct is provided comprising at least two T-DNAmolecules and at least two DNA segments separating said T-DNA molecules,wherein one of the T-DNA molecules contains a selectable marker gene anda first conditional lethal gene; and a second conditional lethal generesides in one DNA segment and a third conditional lethal gene residesin the other DNA segment, the second and third conditional lethal genesbeing heterologous to said first conditional lethal gene.

In still a further aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein one of the T-DNA moleculescontains an agronomic gene of interest and a conditional lethal gene. Inanother aspect of the invention, a DNA construct is provided comprisingat least two T-DNA molecules and at least two DNA segments separatingsaid T-DNA molecules, wherein one of the T-DNA molecules contains anagronomic gene of interest and a first conditional lethal gene; and asecond conditional lethal gene heterologous to said first conditionallethal gene resides in any one DNA segment. In still another aspect ofthe invention, a DNA construct is provided comprising at least two T-DNAmolecules and at least two DNA segments separating said T-DNA molecules,wherein one of the T-DNA molecules contains an agronomic gene ofinterest gene and a first conditional lethal gene; and a secondconditional lethal gene resides in one DNA segment and a thirdconditional lethal gene resides in the other DNA segment and the secondand third conditional lethal genes are heterologous to said firstconditional lethal gene. In an aspect of the invention, the expressionof the conditional lethal gene contained within the T-DNA is preferablydriven by a tissue specific promoter or inducible promoter.

In another aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein the T-DNA molecules eachcontain at least two plant expression cassettes, one plant expressingcassette comprising a conditional lethal gene, the other plantexpression cassette containing a selectable marker gene or agronomicgene of interest expressible in plants.

In another aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein the T-DNA molecules eachcontain at least two plant expression cassettes, one plant expressingcassette comprising a conditional lethal gene, the other plantexpression cassette containing a selectable marker gene or agronomicgene of interest expressible in plants; and the conditional lethal genesare heterologous to each other.

A further aspect of the invention, a DNA construct is providedcomprising at least two T-DNA molecules and at least two DNA segmentsseparating said T-DNA molecules, wherein the T-DNA molecules contain atleast two plant expression cassettes, wherein the first T-DNA moelculecomprises a first plant expression cassette comprising a firstconditional lethal gene and a second plant expression cassettecontaining a selectable marker gene or agronomic gene of interestexpressible in plants; and wherein the second T-DNA molecule comprises athird plant expression cassette comprising a second conditional lethalgene heterologous to said first conditional lethal gene and a fourthplant expression cassette containing a selectable marker gene oragronomic gene of interest expressible in plants; and an additionalconditional lethal gene that is heterologous to the conditional lethalgenes comprising the T-DNA molecules is located in at least one DNAsegment. In a further aspect of the invention, the expression of theT-DNA conditional lethal genes is under the control of a tissue specificor inducible promoter.

A method for enhancing the segregation of transgenes in plants describedherein for producing transgenic plant cells and plants comprising thesteps of:

a) introducing a DNA construct into a plant cell, wherein said DNAconstruct comprises a first DNA molecule comprising a first T-DNAmolecule, and a second DNA molecule comprising a second T-DNA molecule,and a plant expression cassette that expresses a conditional lethal geneproduct is located in a DNA segment positioned between said first T-DNAmolecule and said second T-DNA molecule; and

b) treating said plant cell with a protoxin in amounts sufficient tocause the impairment of the cells when said protoxin is converted to atoxic compound by the expression product of the conditional lethal gene;and

c) regenerating unimpaired plant cells into plants.

In a method for enhancing the segregation of transgenes in plants, thepresent invention further providing a method for producing transgenicplant cells and plants comprising the steps of:

a) introducing a DNA construct into a plant cell, said DNA constructcomprises at least two T-DNA molecules and at least two DNA segments,wherein a plant expression cassette that expresses a conditional lethalgene product is located in any one of said DNA segments; and

b) growing said plant cell into a plant; and

c) collecting seeds from said plant; and

d) planting the seeds to cause germination of the seeds and growth ofseedling plants; and

e) treating the seedling plants with a protoxin in amounts sufficient tocause impairment of cells of the treated seedling plants when saidprotoxin is converted to a toxic compound by the expression product ofthe conditional lethal gene.

The afore described method, wherein the conditional lethal gene productencodes for a protein comprising phosphonate monoester hydrolase,carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithinedeacetylase, P450 monooxygenase CPY105A1, cytosine deaminase,indoleacetamide hydrolase, or β-glucuronidase.

The afore described method, wherein said protoxin comprises phosphonateester of glyphosate, carboxylate ester of glyphosate, glyphosate,N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin,5-fluorocytosine, naphthyl-acetamide, or glucuronic acid conjugates ofherbicides.

The afore described method, wherein said DNA construct is introducedinto a plant cell by an Agrobacterium mediated transformation method.

The afore described method, wherein a first T-DNA molecule and a secondT-DNA molecule further comprise plant expression cassettes encoding fortraits selected from the group consisting of herbicide tolerance,antibiotic resistance, insect resistance, disease resistance, stressresistance, pharmaceutical protein expression, enhanced nutrition, andenhanced yield.

The afore described method, wherein said treated seedling plants thatconvert the protoxin into a toxic compound by the expression product ofthe conditional lethal gene are killed.

A DNA construct is provided wherein a first T-DNA molecule is borderedby a first right border DNA sequence (RB) and a first left border DNAsequence (LB) linked to a second T-DNA molecule that is bordered by asecond right border DNA sequence and a second left border DNA sequence,and the two T-DNA molecules are positioned in the DNA construct to havean orientation with respect to each other consisting of RB-firstT-DNA-LB-LB-second T-DNA-RB, wherein the first RB and second RB arelinked and contained in any one of the T-DNA molecules is at least onefunctional element required for maintenance of the DNA construct as aplasmid in a bacteria cell and a plant selectable marker gene thatprovides antibiotic or herbicide selection resides in this T-DNA.

A method for providing a transgenic plant with independently segregatingtransgenes comprising the steps of: a) introducing a DNA molecule into aplant cell by an Agrobacterium mediated method, wherein said DNAmolecule consists of a first right border region linked to at least onetransgene of agronomic interest linked to a first left border regionlinked to a second left border region linked to a positive selectablemarker transgene linked to a plasmid maintenance element linked to asecond right border region, wherein the second right border region islinked to the first right border region; and b) regenerating said plantcell into a transgenic plant by positive selection provided byexpression of said positive selectable marker transgene; and c)selecting said transgenic plant for the presence of said transgene ofagronomic interest; and d) screening said transgenic plant by a DNAdetection method that identifies the linkage of said transgene ofagronomic interest and said positive selectable marker transgene; and e)growing said transgenic plant into a fertile plant in which saidtransgene of agronomic interest and said positive selectable markertransgene are not linked. The method further comprises the steps of: a)harvesting progeny from said fertile plant; and b) selecting from saidprogeny those individuals that contain said transgene of agronomicinterest and do not contain said positive selectable marker transgene.

A method for providing a transgenic plant with independently segregatingtransgenes comprising the steps of: a) introducing a DNA molecule into aplant cell by an Agrobacterium mediated method, wherein said DNAmolecule consists of an Agrobacterium Ti plasmid first border regionlinked to at least one transgene of agronomic interest linked to anAgrobacterium Ti plasmid second border region linked to a positiveselectable marker transgene; and b) regenerating said plant cell into atransgenic plant by positive selection provided by expression of saidpositive selectable marker transgene; and c) selecting said transgenicplant for the presence of said transgene of agronomic interest; and d)screening said transgenic plant by a DNA detection method thatidentifies the linkage of said transgene of agronomic interest and saidpositive selectable marker transgene; and e) growing said transgenicplant into a fertile plant. The method further comprising the steps of:a) harvesting progeny from said fertile plant; and b) selecting fromsaid progeny those that contain said transgene of agronomic interest anddo not contain said positive selectable marker transgene, wherein saidprogeny are seeds or seedlings thereof. The transgene of agronomicinterest provides an agronomic trait comprising herbicide tolerance,increased yield, insect control, fungal disease resistance, virusresistance, nematode resistance, bacterial disease resistance,mycoplasma disease resistance, modified oils production, high oilproduction, high protein production, germination and seedling growthcontrol, enhanced animal and human nutrition, low raffinose,environmental stress tolerance, increased digestibility, industrialenzyme production, pharmaceutical peptides and small moleculeproduction, improved processing traits, proteins improved flavor,nitrogen fixation, hybrid seed production, reduced allergenicity,biopolymers, or biofuel production. The method further provides atransgenic plant that is a crop plant and a positive selectable markertransgene provides tolerance to an antibiotic or herbicide. The positiveselectable marker transgene is also linked to a plasmid maintenanceelement. The positive selectable marker transgene and plasmidmaintenance element may also be linked to a conditional lethaltransgene.

A method for providing a transgenic plant with independently segregatingtransgenes comprising the steps of: a) introducing a DNA molecule into aplant cell, wherein said DNA molecule consists of an Agrobacterium Tiplasmid first border region linked to at least one transgene ofagronomic interest linked to an Agrobacterium Ti plasmid second borderregion linked to a positive selectable marker transgene; and b)regenerating said plant cell into a transgenic plant by positiveselection provided by expression of said positive selectable markertransgene; and c) selecting said transgenic plant for the presence ofsaid transgene of agronomic interest; and c) growing said transgenicplant into a fertile plant that contains said transgene of agronomicinterest and said positive selectable marker transgene; and e)harvesting seeds from said fertile plant; and f) selecting the seeds orseedlings thereof, that contain said transgene of agronomic interest anddo not contain said positive selectable marker transgene.

A DNA plasmid consisting of a first Agrobacterium Ti plasmid rightborder region linked to at least one transgene of agronomic interestlinked to a second Agrobacterium Ti plasmid right border region linkedto an antibiotic selectable marker transgene or a herbicide selectablemarker transgene wherein said antibiotic selectable marker transgene orherbicide selectable marker transgene is linked to a plasmid maintenanceelement. The antibiotic selectable marker transgene or herbicideselectable marker transgene may also be linked to a conditional lethaltransgene.

A DNA plasmid consisting of a first Agrobacterium Ti plasmid left borderregion linked to at least one transgene of agronomic interest linked toa second Agrobacterium Ti plasmid left border region linked to anantibiotic selectable marker transgene or a herbicide selectable markertransgene wherein said antibiotic selectable marker transgene orherbicide selectable marker transgene is linked to a plasmid maintenanceelement. The antibiotic selectable marker transgene or herbicideselectable marker transgene may also be linked to a conditional lethaltransgene.

A DNA plasmid consisting of an Agrobacterium Ti plasmid right borderregion linked to at least one transgene of agronomic interest linked toan Agrobacterium Ti plasmid left border region linked to an antibioticselectable marker transgene or a herbicide selectable marker transgenewherein said antibiotic selectable marker transgene or herbicideselectable marker transgene is linked to a plasmid maintenance element.The antibiotic selectable marker transgene or herbicide selectablemarker transgene may also be linked to a conditional lethal transgene.

A method of determining the expression pattern of a tissue specific DNApromoter for use in transgenic plants the steps comprising:

b) constructing of a DNA construct containing at least a first and asecond plant expression cassette, wherein the first plant expressioncassette has a constitutive promoter and the second plant expressioncassette has a tissue specific promoter and wherein the plant expressioncassettes are in separate T-DNAs and:

c) transforming the DNA construct into a plant cell; and

d) regenerating said plant cell into a fertile plant; and

e) assaying said plant for the presence of the plant expressioncassettes; and

f) assaying said plant by a DNA detection method that identifies thelinkage of the plant expression cassettes; and

g) assaying said plant for the expression pattern, or expression rate,or expression level of a gene product produced by the expressioncassette having the tissue specific promoter; and

f) selecting said tissue specific promoter for use in the constructionof additional DNA constructs.

In a method for enhancing the segregation of transgenes in plants, thepresent invention further provides a method for stacking transgenetraits in plants comprising the steps of: a) constructing a first DNAconstruct comprising at least two T-DNA molecules, wherein a first T-DNAmolecule comprises a first plant expression cassette that provides atleast one agronomic gene of interest; and a second T-DNA moleculecomprising a second plant expression cassette that provides a positiveselectable marker gene product functional in plant cells and a thirdplant expression cassette that provides a conditional lethal geneproduct functional in plant cells; and b) transforming said first DNAconstruct into a plant cell; and c) treating said plant cell with aneffective amount of a positive selection compound for which tolerance isprovided by said positive selectable marker gene product; and d)regenerating a positive selection compound tolerant plant cell into afertile plant; and e) collecting seeds from said fertile plant; and f)planting seeds from said fertile plant that germinate to produceseedling plants; and g) treating the seedling plants with a protoxin inamounts sufficient to cause damage or death to the plant cells of thetreated seedlings when said protoxin is converted to a toxic compound bysaid conditional lethal gene product; and h) selecting a seedling plantfrom the treated seedling plants for the presence of the first T-DNA andfor no plant cell impairment of the type caused by the conversion of theprotoxin to a toxic compound; and i) propagating said seedling plantselected in step (h) into a fertile plant; and j) constructing a secondDNA construct comprising at least two T-DNA molecules, wherein a thirdT-DNA molecule comprises a fourth plant expression cassette thatprovides at least one agronomic gene of interest; and the second T-DNAmolecule of the first DNA construct; and k) retransforming said fertileplant of step (i) with said second DNA construct; and l) repeating stepsc-g; and m) selecting a seedling plant for the presence of the agronomicgene of interest of the first DNA construct and the agronomic gene ofinterest of the second DNA construct and for no plant cell impairment ofthe type caused by the conversion of the protoxin to a toxic compound;and n) propagating said seedling plant selected in step (m) into afertile plant.

In a method for enhancing the segregation of transgenes in plants, thepresent invention further provides a method of controlling thedissemination of transgenes by pollen outcrossing comprising the stepsof: a) constructing a DNA construct comprising a first T-DNA moleculeand a second T-DNA molecule, said first T-DNA molecule comprising afirst plant expression cassette and a second plant expression cassette,wherein said first plant expression cassette provides a selectable geneproduct and said second plant expression cassette provides a firstconditional lethal gene product expressed under the control of a plantmale reproductive tissue specific promoter; and said second T-DNAsegment comprising a third plant expression cassette and a fourth plantexpression cassette, wherein said third plant expression cassetteprovides an agronomic gene of interest and said fourth plant expressioncassette provides a second conditional lethal gene product expressedunder the control of a plant pollen specific promoter, wherein saidsecond conditional lethal gene product is heterologous to said firstconditional lethal gene product; and

b) transforming said DNA vector into a plant cell; and

c) growing said plant cell into a fertile first parent plant homozygousfor said first and second T-DNA segments; and

d) crossing said fertile first parent plant with a fertile second parentplant to produce a hybrid plant; and

e) harvesting seed from said hybrid plant; and

f) planting said seed from said hybrid plant in a field under conditionsthat cause the germination of said seed and growth into plants; and

h) treating the plants of step (f) with an effective amount of acompound that is converted to a toxin by the conditional lethal geneproduct expressed under the control of a plant male tissue specificpromoter to cause impairment or death of pollen expressing theconditional lethal gene product.

A method for enhancing the segregation of transgenes in plantscomprising the steps of:

a) introducing at least two DNA constructs into a plant cell, wherein afirst and second DNA construct comprises at least one T-DNA molecule andat least one DNA segment, wherein a plant expression cassette thatexpresses a conditional lethal gene product is located in the DNAsegment; and

b) growing said plant cell into a plant; and

c) collecting seeds from said plant; and

d) planting the seeds to cause germination of the seeds and growth ofseedling plants; and

e) treating the seedling plants with a protoxin in amounts sufficient tocause death of cells of the treated seedling plants when said protoxinis converted to a toxic compound by the expression product of theconditional lethal gene.

The afore described method, wherein the conditional lethal gene productencodes for a protein comprising phosphonate monoester hydrolase,carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithinedeacetylase, P450 monooxygenase CPY105A1, cytosine deaminase,indoleacetamide hydrolase, or β-glucuronidase The afore describedmethod, wherein said protoxin comprises phosphonate ester of glyphosate,carboxylate ester of glyphosate, glyphosate,N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin,5-fluorocytosine, naphthyl-acetamide, or glucuronic acid conjugates ofherbicides.

The afore described method, wherein said DNA construct is introducedinto a plant cell by an Agrobacterium mediated transformation method andthe Agrobacteria comprise nopaline and octopine strains.

The present invention provides DNA constructs that express a conditionallethal gene product comprising phosphonate monoester hydrolase,glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithinedeacetylase, P450 monooxygenase CPY105A1, cytosine deaminase,indoleacetamide hydrolase, or β-glucuronidase, wherein the gene productinteracts with a compound that is converted to a toxin by theconditional lethal gene product, the compound comprising phosphonateester of glyphosate, glyphosate, carboxylate ester of glyphosate,N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin,5-fluorocytosine, naphthyl-acetamide, glucuronic acid conjugates ofherbicides, respectively.

The present invention provides DNA constructs that contain a herbicideresistant gene product comprising genes for class II EPSPS inhibitorresistant enzymes, class I EPSPS inhibitor resistant enzymes,aceto-lactone synthase inhibitor resistant enzymes, glutamine synthaseinhibitor enzymes.

The present invention further comprises DNA constructs that containadditional agronomic genes of interest selected from the groupconsisting of herbicide tolerance, antibiotic resistance, insectresistance, disease resistance, stress resistance, pharmaceuticalprotein expression, enhanced nutrition, and enhanced yield.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Plasmid map of pMON42061

FIG. 2. Plasmid map of pMON51652

FIG. 3. Plasmid map of pMON51653

FIG. 4. Plasmid map of pMON54237

FIG. 5. Plasmid map of pMON51658

FIG. 6. Plasmid map of pMON51661

FIG. 7. Plasmid map of pMON51676

FIG. 8. Plasmid map of pMON42073

FIG. 9. Plasmid map of pMON42070

FIG. 10. Plasmid map of pMON42071

FIG. 11. Plasmid map of pMON42072

FIG. 12. Plasmid map of pMON9443

FIG. 13. Plasmid map of pMON9430

FIG. 14. Plasmid map of pMON17164

FIG. 15. Plasmid map of pMON73105

FIG. 16. Plasmid map of pMON73107

FIG. 17. Plasmid map of pMON73106

FIG. 18. Plasmid map of pMON73108

FIG. 19. Plasmid map of pMON62400

FIG. 20. Plasmid map of pMON65178

FIG. 21. Plasmid map of pMON65179

FIG. 22. Plasmid map of pMON65180

FIG. 23. Plasmid map of pLagILB01

FIG. 24. Plasmid design with duplicated border regions

FIG. 25. Alternative plasmid designs with duplicated border regions

FIG. 26. Plasmid design with two border regions

DETAILED DESCRIPTION OF THE INVENTION

Herein we describe and exemplify compositions and methods that areuseful for enhancing the occurrence of segregating agronomic genes ofinterest transformed into plants as transgenes, comprising T-DNAmolecules that include, but not limited to, herbicide resistance genes;insecticidal protein genes from Bacillus species and other bacteria,fungi, and plants; antibiotic protein genes from viruses, bacteria,fungi, plants and animals; genes affecting plant growth and development,such as genes involved in plant hormone biosynthesis or degradation,vitamin biosynthesis, and cellular architecture, comprising herbicidetolerance, increased yield, insect control, fungal disease resistance,virus resistance, nematode resistance, bacterial disease resistance,mycoplasma disease resistance, modified oils production, high oilproduction, high protein production, germination and seedling growthcontrol, enhanced animal and human nutrition, low raffinose,environmental stress tolerance, increased digestibility, industrialenzyme production, pharmaceutical peptides and small moleculeproduction, improved processing traits, proteins improved flavor,nitrogen fixation, hybrid seed production, reduced allergenicity,biopolymers, or biofuel production.

The DNA constructs of the present invention comprises at least two T-DNAmolecules and at least two Agrobacterium Ti plasmid border regions. In amethod of the present invention steps comprise the treatment of plantcells transformed with the DNA constructs, plants or segregating progenythereof derived from the transformed plant cells, with a protoxin thatwhen converted into a toxin by any one of the conditional lethal geneproducts described herein, eliminates the cells or progeny plants fromthe population of treated cells or plants.

Additionally, it may be desirable to reduce or eliminate a T-DNAmolecule in a particular cell, tissue, or organ, but not in other partsof a plant in which the transgene is expressed. The elimination ofpollen that is expressing insecticidal proteins is desirable as well aseliminating the potential for the outcrossing of transgenes to relatedplants and into adjacent fields from hybrid crops. In this aspect of theinvention, a method is provided applicable to hybrid plants includingmonocotyledons such as rice, wheat, oats, barley, corn and the like, aswell as dicotyledons such as alfalfa, canola, carrot, cotton, oil seedrape, sugar beet, sunflower, soybean, tomato and the like. Generally,this process comprises applying a selected protoxin compound to thetransgenic hybrid crop plant that is expressing a conditional lethalgene product by a male specific promoter, thereby causing the death ofpollen where the expression of the enzyme converts the protoxin into atoxin. Any agronomic gene(s) of interest associated with the allelescontributed by the transgenic parent that carry the conditional lethalgene will be eliminated. In a related method, there is provided aprocess to eliminate a certain T-DNA molecule and the expressioncassettes contained therein from a plant breeding program in which ithas become desirable to remove a particular transgene from the breedingplant parent populations. Such compositions and methods may be used withrespect to any plant that can be genetically modified by biotechnology.

Additionally, the two T-DNAs integrated at physically and geneticallydistinct locations in the plant genome reduce the interactions that canoccur between closely linked genetic elements in a plant expressioncassette. Strong constitutive promoters may influence the expressionpattern or rate of transcription of a closely associated transgenictissue specific promoter. The segregation of the T-DNAs by the methodsof the present invention can reduce the promoter/promoter interactionsthat can be problematic when multiple expression cassettes are used in asingle T-DNA. The methods of the present invention can be applied todetermine if transgene plant expression cassettes interact to influencethe expression pattern of the genes of interest, and as a result, thecombinations of expression cassettes can be optimized to enable a morepredictive pattern of expression of the transgenes. The use of themethod for this purpose saves considerable time and expense in designingand making DNA constructs and in producing and evaluating transgenicplants.

The methods of the present invention provide enhanced planttransformation efficiency, wherein the efficiency is measured in thetransformed plants as a high frequency of expression of both T-DNAinserts from a two T-DNA construct and the T-DNA inserts are located atunlinked genetic loci and the transformed plants have a low occurrenceof extraneous vector DNA in the plant genome. In these methods, the DNAconstruct comprising a two T-DNA construct, have T-DNA border sequencesthat form an orientation dependent enhancement within the DNA construct,the orientation being a right border-first T-DNA-left border, leftborder-second T-DNA-right border.

In a further aspect of the method, at least two DNA constructs eachcomprising at least one T-DNA and a DNA segment containing a conditionallethal gene are introduced into disarmed Agrobacteria cells, one DNAconstruct into a nopaline Agrobacterium strain and one DNA constructinto an octopine Agrobacterium strain. The two strains, each containinga different DNA construct, are mixed together in an Agrobacteriummediated plant transformation method.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York (1991); and Lewin,Genes V, Oxford University Press: New York (1994). The nomenclature forDNA bases as set forth at 37 CFR § 1.822 is used. The standard one- andthree-letter nomenclature for amino acid residues is used.

Abbreviations for nucleotide bases in nucleic acid codes as used hereinare: A=adenosine; C=cytosine; G=guanosine; T=thymidine. Codes used forsynthesis of oligonucleotides as used herein are: N=equimolar A, C, G,and T; I=deoxyinosine; K=equimolar G and T; R=equimolar A and G;S=equimolar C and G; W=equimolar A and T; Y=equimolar C and T.

“Carboxylate ester of glyphosate” comprise a class of protoxin compoundsthat can be converted to active glyphosate by hydrolysis of the esterbond between the carboxy group and N-phosphonomethyl-glycine.

“conditional lethal gene product” as defined herein, as a peptide,protein or enzyme that when expressed in transgenic plant cells convertsa chemical compound that has low phytotoxicity or is nonphytotoxic inthe present environment of the plant cell into a compound that is highlytoxic in a plant cell environment that now includes the conditionallethal gene product. Examples of conditional lethal gene productsinclude, but are not limited to phosphonate monoester hydrolase,carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithinedeacetylase, P450 monooxygenase CPY105A1 or β-glucuronidase. Thisdefinition includes a herbicide tolerant plant cell environment in whichthe herbicide is converted into a compound for which the plant cell isno longer tolerant.

As used herein, the term “comprise” can be used interchangeably with thephrase “includes, but is not limited to.”

“CP4”, “aroA:CP4”, “AGRTU.aroA:CP4”, “CP4 EPSPS” and “EPSPS CP4” referto the EPSP synthase gene or protein purified from Agrobacteriumtumefaciens (AGRTU) strain CP4 that when expressed in plants conferstolerance to glyphosate and glyphosate containing herbicide formulations(U.S. Pat. No. 5,633,435, herein incorporated by reference in it'sentirety). The gene sequence maybe native or modified for enhancedexpression in plants.

A DNA “segment” refers a region of DNA sequence of a DNA construct forwhich it is generally not desirable to have it integrated into a plantgenome. A DNA segment is between or flanks the T-DNA molecules. A DNAsegment often contains the genetic elements for replication of plasmidsin bacteria and is the DNA sequence that various elements and expressioncassettes of the present invention are located.

“GOX” refers to glyphosate oxidoreductase protein and the gene thatencodes for enzymes that can degrade glyphosate(N-phosphonomethyl-glycine) and other substrates toaminomethylphosphonate (AMPA). The glyphosate oxidoreductase genenucleotide sequence and modifications made to the nucleotide sequence asdescribed in U.S. Pat. No. 5,463,175 and U.S. Pat. No. 5,312,910, hereinincorporated by reference in their entirety.

A “homolog” of a gene of one species is a nucleic acid sequence to whicha probe or primer derived from the gene that binds under at leastmoderately stringent hybridization conditions to a nucleic acid sequenceof a second species.

“Impairment” of a plant cell or a plant by the presence of a toxinmolecule refers to a reduction in the normal function of a plant cell ora plant. The impairment can be determined by a visual observation or bymeasuring a physiological or chemical change in the plant cell or plant.Impairment in the extreme results in death of the plant cell, planttissues or plants.

An “isolated” nucleic acid is substantially separated or purified awayfrom other nucleic acid sequences in the cell of the organism in whichthe nucleic acid naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA, by conventional nucleic acid-purificationmethods. The term also embraces recombinant nucleic acids and chemicallysynthesized nucleic acids.

The term “chimeric” refers to the product of the fusion of portions oftwo or more different nucleic acids or proteins.

For the purposes of the present invention, the term “glyphosate”includes any herbicidally active form of N-phosphonomethylglycine(including any salt thereof) and other forms that result in theproduction of the glyphosate anion in plants. Glyphosate is the activeingredient of Roundup® and Roundup Ultra® herbicide formulations, forexample (Monsanto Company, St. Louis, Mo.). Treatments with “glyphosate”refer to treatments with the Roundup® or Roundup Ultra® herbicideformulation, unless otherwise stated. Plant transformation andregeneration in tissue culture use glyphosate or salts of glyphosate.Whole plant assays use formulated Roundup® Ultra. Additionalformulations with herbicide activity that containN-phosphonomethylglycine or any of its salts are herein included as aglyphosate herbicide.

“Glycerol glyphosate” refers to the compound glycine, N-([hydroxy (2,3dihydroxy propoxy)phosphonyl]methyl) is representative of the generalclass of phosphonate esters of glyphosate that is a substrate forphosphonate monoester hydrolases, is relatively non-cytotoxic andtranslocates to developing plant tissues.

The term “glyphosate resistance gene” refers to any gene that, whenexpressed as a transgene in a plant, confers the ability to toleratelevels of the herbicide glyphosate that would otherwise damage or killthe plant. Any glyphosate tolerance gene known to the skilled individualare suitable for use in the practice of the present invention.Glyphosate inhibits the shikimic acid pathway that leads to thebiosynthesis of aromatic compounds including amino acids, plant hormonesand vitamins. Specifically, glyphosate inhibits the enzyme5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A variety of nativeand variant EPSPS enzymes have been expressed in transgenic plants inorder to confer glyphosate tolerance (Singh et al., In “Biosynthesis andMolecular Regulation of Amino Acids in Plants”, Amer. Soc. Plant Phys.,1992), any of which can be used in the invention. Examples of some ofthese EPSPSs include those described and/or isolated in accordance withU.S. Pat. No. 4,940,835, U.S. Pat. No. 4,971,908, U.S. Pat. No.5,145,783, U.S. Pat. No. 5,188,642, and U.S. Pat. No. 5,310,667, hereinincorporated by reference in their entirety. They can also be derivedfrom a structurally distinct class of non-homologous EPSPS genes, suchas the class II EPSPS genes isolated from Agrobacterium sp. strain CP4(AGRTU.aroA:CP4) as described in U.S. Pat. Nos. 5,633,435, 5,804,425 and5,627,061, herein incorporated by reference in their entirety.

The term “native” refers to a naturally-occurring (“wild-type”) nucleicacid or polypeptide.

A first nucleic-acid sequence is “operably” connected or “linked” with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to aprotein-coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein-codingregions, are in the same reading frame.

The enzyme “phosphonate monoester hydrolase” (PEH) of the presentinvention catalyzes the cleavage of phosphonate esters into the freephosphonate and an alcohol. The enzyme was first identified by itsability to hydrolyze the glyceryl glyphosate into glycerol andglyphosate. The enzyme has since been shown to hydrolyze other protoxincompounds as well as numerous other phosphonate monoesters includingcolorimetric substrates. The gene (pehA) encoding the subjectphosphonate monoester hydrolase enzyme is directed to the isolation ofsuch a gene from Pseudomonas caryophilli PG2982 (NCIMB #12533) asdescribed in U.S. Pat. No. 5,254,801, herein incorporated by referencein its' entirety.

The term “plant” encompasses any higher plant and progeny thereof,including monocots (e.g., lily, corn, rice, wheat, barley, etc.), dicots(e.g., tomato, potato, soybean, cotton, canola, tobacco, etc.), andincludes parts of plants, including reproductive units of a plant (e.g.,seeds), fruit, flowers, etc.

A “recombinant” nucleic acid is made by an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

A “reproductive unit” or propagule of a plant is any totipotent part ortissue of the plant from which one can obtain a progeny of the plant,including, for example, seeds, cuttings, tubers, buds, bulbs, somaticembryos, cultured cells (e.g., callus or suspension cultures), etc.

The terms “DNA construct” or “DNA vector” refers to any plasmid, cosmid,virus, autonomously replicating sequence, phage, or other circularsingle-stranded or double-stranded DNA or RNA derived from any sourcethat includes one or more DNA sequences, such as promoters,protein-coding sequences, 3′ untranslated regions, etc., that have beenlinked in a functionally operative manner by recombinant DNA techniques.Recombinant DNA vectors for plant transformation are commonlydouble-stranded circular plasmids capable of replication in a bacterialcell. Conventional compositions and methods for making and usingrecombinant nucleic acid constructs are discussed, inter alia, inMolecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrooket al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989; and Current Protocols in Molecular Biology, ed. Ausubel et al.,Greene Publishing and Wiley-Interscience, New York, 1992 (with periodicupdates). See also, e.g., Mailga et al., Methods in Plant MolecularBiology, Cold Spring Harbor Press (1995); Birren et al., Genome AnalysisDetecting Genes, 1, Cold Spring Harbor, N.Y. (1998); Birren et al.,Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, N.Y. (1998); andClark et al., Plant Molecular Biology: A Laboratory Manual, Springer,New York (1997).

Methods for chemical synthesis of nucleic acids are discussed, forexample, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862 (1981),and Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981). Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers.

A number of vectors suitable for stable transformation of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual (1985, supp. 1987);Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress (1989); and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers (1990). Typically, plant expression vectors includeone or more transcription units, each of which includes: a 5′untranslated region, which includes sequences that control transcription(e.g., cis-acting promoter sequences such as enhancers, thetranscription initiation start site, etc.) and translation (e.g., aribosome binding site) of an operably linked protein-coding sequence(“open reading frame”, ORF); a 3′ untranslated region that includesadditional regulatory regions from the 3′ end of plant genes (Thornburget al., Proc. Natl. Acad. Sci. USA 84:744 (1987); An et al., Plant Cell1:115 (1989), e.g., a 3′ terminator region to increase mRNA stability.In addition, such constructs commonly include a selectable or screenablemarker and optionally an origin of replication or other sequencesrequired for replication of the vector in a host cell.

Plant expression vectors optionally include RNA processing signals,e.g., introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. An intron element isidentified in a description of an expression cassette by “I-” precedinga gene name, coding sequence name or genomic identification number. Inaddition, the expression vectors may also include additional regulatorysequences from the 3′-untranslated region of plant genes. These 3′untranslated regions contain mRNA transcription termination signals,thus these regions when used in chimeric expression cassettes aredesignated with “T-” followed by a gene name, coding sequence name orgenomic identification number. Other movable elements contained in plantexpression vectors may include 5′ leader sequences, designated by “L-”and transit signal sequences designated by “TS-”, each followed by agene name, coding sequence name or genomic identification number. Theelements of a plant expression cassette are described in a 5′ to 3′orientation of the linked elements using the element names separated bya “/”.

The term “promoter” or “promoter region” refers to a nucleic acidsequence, usually found upstream (5′) to a coding sequence, thatcontrols expression of the coding sequence by controlling production ofmessenger RNA (mRNA) by providing the recognition site for RNApolymerase and/or other factors necessary for start of transcription atthe correct site. As contemplated herein, a promoter or promoter regionincludes variations of promoters derived by means of ligation to variousregulatory sequences, random or controlled mutagenesis, and addition orduplication of enhancer sequences. The promoter region disclosed herein,and biologically functional equivalents thereof, are responsible fordriving the transcription of coding sequences under their control whenintroduced into a host as part of a suitable recombinant vector, asdemonstrated by its ability to produce mRNA. A “P-” preceding a gene,coding sequence name or genomic identification number designates theelement as a promoter. The genus species of the source of the promoterelement is used in the promoter name, for example, Zea mays (corn) isabbreviated to Zm, Cauliflower mosaic virus is CaMV, Triticum aesativum(wheat) is Ta, Orysae sativa (rice) is Os and so forth. The 35S promoterof CaMV is therefore, P-CaMV.35S.

“Regeneration” refers to the process of growing a plant from a plantcell (e.g., plant protoplast or explant).

“Reporter” refers to a gene and corresponding gene product that whenexpressed in transgenic organisms produces a product detectable bychemical or molecular methods or produces an observable phenotype.

“Restriction enzyme” refers to an enzyme that recognizes a specificpalindromic sequence of nucleotides in double stranded DNA and cleavesboth strands; also called a restriction endonuclease. Cleavage typicallyoccurs within the restriction site.

“Retransformation” refers to a method, wherein a new transgene isintroduced by the methods of plant transformation into a plant cell thatin itself is a transgenic cell produced by transformation at an earliertime.

“Selectable marker” refers to a nucleic acid sequence whose expressionconfers a phenotype facilitating identification of cells containing thenucleic acid sequence. Selectable markers include those that conferresistance to toxic chemicals (e.g. ampicillin resistance, kanamycinresistance), complement a nutritional deficiency (e.g. uracil,histidine, leucine), or impart a visually distinguishing characteristic(e.g. color changes or fluorescence). Useful dominant selectable markergenes include genes encoding antibiotic resistance genes (e.g.,resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin orspectinomycin); and herbicide resistance genes (e.g., phosphinothricinacetyltransferase, modified ALS, modified class I EPSPSs, class IIEPSPSs). A useful strategy for selection of transformants for herbicideresistance is described, e.g., in Vasil, Cell Culture and Somatic CellGenetics of Plants, Vols. I-III, Laboratory Procedures and TheirApplications Academic Press, New York (1984).

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under given hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

“T-DNA molecule” refers to a DNA molecule that integrates into a plantgenome via an Agrobacterium mediated transformation method. The ends ofthe T-DNA molecule are defined in the present invention as being flankedby the border regions of the T-DNA from Agrobacterium Ti plasmids. Theseborder regions are generally referred to as the Right border and Leftborder regions and exist as variations in nucleotide sequence and lengthdepending on whether they are derived from nopaline or octopineproducing strains of Agrobacterium. The border regions commonly used inDNA constructs designed for transferring transgenes into plants areoften several hundred polynucleotides in length and comprise a nick sitewhere an endonuclease digests the DNA to provide a site for insertioninto the genome of a plant. The T-DNA molecule generally contain one ormore plant expression cassettes.

“Tolerant” refers to a reduced toxic effect of a herbicide on the growthand development of microorganisms and plants.

“Transcription” refers to the process of producing an RNA copy from aDNA template.

“Transformation” refers to a process of introducing an exogenous nucleicacid sequence (e.g., a vector, recombinant nucleic acid molecule) into acell or protoplast that exogenous nucleic acid is incorporated into achromosome or is capable of autonomous replication.

“Transformed” or “transgenic” refers to a cell, tissue, organ, ororganism into that has been introduced a foreign nucleic acid, such as arecombinant vector. A “transgenic” or “transformed” cell or organismalso includes progeny of the cell or organism and progeny produced froma breeding program employing such a “transgenic” plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe foreign nucleic acid.

The term “transgene” refers to any nucleic acid sequence normative to acell or organism transformed into said cell or organism. “Transgene”also encompasses the component parts of a native plant gene modified byinsertion of a normative nucleic acid sequence by directedrecombination.

The term “translation” refers to the production the corresponding geneproduct, i.e., a peptide, polypeptide, or protein from a mRNA.

“Isolated,” “Purified,” “Homogeneous” Polypeptides. A polypeptide is“isolated” if it has been separated from the cellular components(nucleic acids, lipids, carbohydrates, and other polypeptides) thatnaturally accompany it or that is chemically synthesized or recombinant.A monomeric polypeptide is isolated when at least 60% by weight of asample is composed of the polypeptide, preferably 90% or more, morepreferably 95% or more, and most preferably more than 99%. Proteinpurity or homogeneity is indicated, for example, by polyacrylamide gelelectrophoresis of a protein sample, followed by visualization of asingle polypeptide band upon staining the polyacrylamide gel; highpressure liquid chromatography; or other conventional methods. Coatproteins can be purified by any of the means known in the art, forexample as described in Guide to Protein Purification, ed. Deutscher,Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, ProteinPurification: Principles and Practice, Springer Verlag, New York, 1982.

There are a variety of conventional methods and reagents for “labeling”polypeptides and fragments thereof. Typical labels include radioactiveisotopes, ligands or ligand receptors, fluorophores, cherniluminescentagents, and enzymes. Methods for labeling and guidance in the choice oflabels appropriate for various purposes are discussed, e.g., in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989) and Ausubel et al., Greene Publishing and Wiley-Interscience, NewYork, (1992).

Mature protein coding region. This term refers to the sequence of apost-translationally processed protein product, for example, EPSPsynthase remaining after the chloroplast transit peptide sequence hasbeen removed.

Polypeptide fragments. The present invention also encompasses fragmentsof a coding sequence that lacks at least one residue of a nativefull-length protein, but that specifically maintains functional activityof the protein.

Transit peptide or targeting sequence (TS-). These terms generally referto peptide sequences that when linked to a protein of interest directsthe protein to a particular tissue, cell, subcellular location, or cellorganelle. Examples include, but are not limited to, chloroplast transitpeptides, nuclear targeting signals, and vacuolar signals. Thechloroplast transit peptide is of particular utility in the presentinvention to direct expression of the EPSPS enzyme to the chloroplast.

The term “plant” encompasses any higher plant and progeny thereof,including monocots (e.g., corn, rice, wheat, barley, etc.), dicots(e.g., soybean, cotton, tomato, canola, potato, Arabidopsis, tobacco,etc.), gymnosperms (pines, firs, cedars, etc.) and includes parts ofplants, including reproductive units of a plant (e.g., seeds, bulbs,tubers, or other parts or tissues from that the plant can bereproduced), fruit, flowers, etc.

Included within the terms “scorable marker genes” are also genes thatencode a secretable marker whose secretion can be detected as a means ofidentifying or selecting for transformed cells. Examples include markersthat encode a secretable antigen that can be identified by antibodyinteraction, or even secretable enzymes that can be detectedcatalytically. Secretable proteins fall into a number of classes,including small, diffusible proteins that are detectable, (e.g., byELISA), small active enzymes that are detectable in extracellularsolution (e.g., α-amylase, β-lactamase, phosphinothricinacetyltransferase), or proteins that are inserted or trapped in the cellwall (such as proteins that include a leader sequence such as that foundin the expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

A vector or construct may also include various regulatory elements. The5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the DNA moleculeof the present invention, and can be specifically modified if desired soas to increase translation of mRNA. For a review of optimizingexpression of transgenes, see Koziel et al., (Plant Mol. Biol.32:393-405 (1996). The 5′ non-translated regions can also be obtainedfrom plant viral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maizedwarf mosaic virus, Alfalfa mosaic virus, among others) from suitableeukaryotic genes, plant genes (wheat and maize chlorophyll a/b bindingprotein gene leader), or from a synthetic gene sequence. The presentinvention is not limited to constructs wherein the non-translated regionis derived from the 5′ non-translated sequence that accompanies thepromoter sequence. The leader sequence could also be derived from anunrelated promoter or coding sequence. Leader sequences useful incontext of the present invention comprise the maize Hsp70 leader (U.S.Pat. No. 5,362,865 and U.S. Pat. No. 5,859,347, herein incorporated byreference in their entirety.), and the TMV omega element (Gallie et al.,The Plant Cell 1:301-311 (1989). Intron sequences are known in the artto aid in the expression of transgenes in monocot plant cells. Examplesof such introns include the Adh intron 1 (Callis et al., Genes andDevelop. 1:1183-1200 (1987), the sucrose synthase intron (Vasil et al.,Plant Physiol. 91:1575-1579 (1989), U.S. Pat. No. 5,955,330), firstintron of the rice actin gene (U.S. Pat. No. 5,641,876), hereinincorporated by reference in their entirety.

A vector may also include a transit peptide nucleic acid sequence (TS-).The glyphosate target in plants, the 5-enolpyruvyl-shikimate-3-phosatesynthase (EPSPS) enzyme, is located in the chloroplast. Manychloroplast-localized proteins, including EPSPS, are expressed fromnuclear genes as precursors and are targeted to the chloroplast by achloroplast transit peptide (CTP) that is removed during the importsteps. Examples of other such chloroplast proteins include the smallsubunit (SSU) of Ribulose-1,5,-bisphosphate carboxylase, Ferredoxin,Ferredoxin oxidoreductase, the light-harvesting complex protein I andprotein II, and Thioredoxin F. It has been demonstrated in vivo and invitro that non-chloroplast proteins may be targeted to the chloroplastby use of protein fusions with a CTP and that a CTP sequence issufficient to target a protein to the chloroplast. Incorporation of asuitable chloroplast transit peptide, such as, the Arabidopsis thaliana(At.) EPSPS CTP (Klee et al., Mol. Gen. Genet. 210:437-442), and thePetunia hybrida (Ph.) EPSPS CTP (della-Cioppa et al., Proc. Natl. Acad.Sci. USA 83:6873-6877) has been shown to target heterologous EPSPSprotein sequences to chloroplasts in transgenic plants. The productionof glyphosate tolerant plants by expression of a fusion proteincomprising an amino-terminal CTP with a glyphosate resistant EPSPSenzyme is well known by those skilled in the art, (U.S. Pat. No.5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571,EP 189707, EP 508909, and EP 924299, herein incorporated by referencedin their entirety). Those skilled in the art will recognize thatvarious chimeric constructs can be made that utilize the functionalityof a particular CTP to import conditional lethal gene products into theplant cell chloroplast when necessary to provide the product there forefficacy of the phenotype.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to the gene ofinterest. The 3′ non-translated region of a recombinant DNA moleculecontains a polyadenylation signal that functions in plants to cause theaddition of adenylate nucleotides to the 3′ end of the RNA. The 3′non-translated region can be obtained from various genes that areexpressed in plant cells. The nopaline synthase 3′ untranslated region(Fraley et al., Proc. Natl. Acad. Sci. 80:4803-4807 (1983), the 3′untranslated region from pea small subunit Rubisco gene (Coruzzi et al.,EMBO J. 3:1671-1679, 1994), the 3′ untranslated region from soybean 7Sseed storage protein gene (Schuler et al., Nuc Acids Res. 10:8225-8244,1982) are commonly used in this capacity. The 3′ transcribed,non-translated regions containing the polyadenylate signal ofAgrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

For embodiments of the invention in which the use of a constitutivepromoter is desirable, any well-know constitutive plant promoter may beused. Constitutive plant promoters include, for example, the cauliflowermosaic virus (CaMV) ³⁵S promoter, which confers constitutive, high-levelexpression in most plant tissues (see, e.g., Odel et al., Nature313:810, 1985), including monocots (see, e.g., Dekeyser et al., PlantCell 2:591, 1990); Terada et al., Mol. Gen. Genet. 220:389, 1990); thenopaline synthase promoter (An et al., Plant Physiol. 88:547, 1988), theoctopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989),cauliflower mosaic virus 19S promoter, figwort mosaic virus ³⁵Spromoter, sugarcane bacilliform virus promoter, commelina yellow mottlevirus promoter, rice cytosolic triosephosphate isomerase promoter,adenine phosphoribosyltransferae promoter, rice actin 1 promoter,mannopine synthase promoter, histone promoter, and a tobaccoconstitutive promoter as disclosed in U.S. Pat. No. 5,824,872.

For other embodiments of the invention, well-known plant gene promotersthat are regulated in response to environmental, hormonal, chemical,and/or developmental signals may be used, including promoters regulatedby (1) heat (Callis et al., Plant Physiol. 88:965, 1988), (2) light(e.g., pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell 1:471 (1989);maize rbcS promoter, Schaffner and Sheen, Plant Cell 3:997 (1991); orchlorophyll a/b-binding protein promoter, Simpson et al., EMBO J.4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al.,Plant Cell 1:969, 1989), (4) wounding (e.g., wunI, Siebertz et al.,Plant Cell 1:961, 1989); or (5) chemicals such as methyl jasmonate,salicylic acid, etc. It may also be advantageous to employ (6)organ-specific promoters (e.g., Roshal et al., EMBO J. 6:1155 (1987);Schernthaner et al., EMBO J. 7:1249, 1988); Bustos et al., Plant Cell1:839, 1989).

It is desirable in one aspect of the invention to use promoters that arepreferentially expressed in a reproductive tissue. Any well-known male-or female-specific plant promoter may be used. Promoters that arepreferentially expressed in a male tissue, include, but are not limitedto, the following: the Xyl promoter (Bih et al., J. Biol. Chem.274:2884-2894, 1999), RA8 (Jeon et al., Plant Mol. Biol. 39:3544, 1999),Ms45 (WO 9859061), SGB6 (U.S. Pat. No. 5,837,850), Tap1 (WO 9827201),Osg6B (Matsuda et al., Plant Biotechnol. (Tokyo) 14:157-161 (1997),Sta44 (CA 2165934), MS2 (Aarts et al., Plant J. 12:615-623, 1997),Zmg13, TA29 (WO 9325695), SLG and SLR1 (WO 9425613), RST2 (WO 9713401),ZmC5 (WO 9942587), and A3, A6, A9 (WO 9302197, U.S. Pat. No. 5,723,754)promoters, the rice YY1 and YY2 anther-specific promoters (Hihara etal., Plant Mol. Biol. 30:1181-1193 (1996), the corn pollen-specificpromoters ZmABP1 and ZmABP2 (Lopez et al., Proc. Nat. Acad. Sci. USA93:7415-7420 (1996), the tapetum-specific oleosin-like gene promoters inbrassica (Ross et al., Plant J. 9:625-637 (1996), the pollen-specificDEFH125 gene promoter from Antirrhinum (Zachgo et al., Plant J. 11:1043-1050 (1997), the pollen-specific LePro 1 promoter (Yu et al., PlantMol. Biol. 36:699-707 (1998), the anther-specific MROS gene promoters(Matsunaga et al., Plant J. 10:679-689 (1990), the pollen-specificpolygalaturonase gene promoter from brassica (U.S. Pat. No. 5,689,053)and maize (U.S. Pat. No. 5,412,085), the pollen-specific Lat52 and Lat59promoters (Twell et al., Development 109:705-713 (1990), theanther-specific 1,3-beta-glucanase gene promoter (U.S. Pat. No.5,955,653), and the Zea mays profilin multigene family anther and pollenpromoters (Staiger et al., Plant J. 4:631-641 (1993). Promoter hybridscan be made that combine the functions of pollen, anther, tapetal celland other male tissue specific expression into a single DNA molecule,for example, a fusion of the DNA sequences of the Osg6B promoter fromrice and the P-Zm.Tas9 promoter (an element isolated from a corn tasselgenomic library wherein the Zm.Tas9 coding sequence has homology to Zeamays profilin coding sequences) to create P-Os.Osg6B-Zm.Tas9 where theTATA box of the 5′ Osg6B promoter sequence is modified or deleted toprevent transcription from that element. An additional promoter elementcan be combined with the rice-corn male promoter by the same method, forexample, the wheat P-Ta.1674-19 promoter isolated from wheat sporophylltissue, this triple promoter fusion provides broad spectrum monocot anddicot male tissue expression. The resulting promoter,P-Os.Osg6B-Zm.Tas9-Ta.1674-19 can be combined with the regulatory RNAbinding protein coding sequences of the present invention to enable highlevels of protein expression in the male tissues. These hybrid promotersare useful for providing expression at all stages of male tissuedevelopment.

Promoters that are preferentially expressed in a female tissue of theplant, include, but not limited to, the following promoters: the styleand stigma specific promoters (EP 412006) and S-locus specificglycoprotein gene promoters; P26, P19, B200i4-2 (WO 9839462); DefH9 (WO9828430); cysteine-rich extension-like protein gene promoters (Goldmanet al., Plant Cell 4:1041-1051, 1992); ovule-specific 039, 0126, 0108and 0141 gene promoters from orchid (Nadeau et al., Plant Cell8:213-239, 1996), the potato pistil-specific SK2 gene promoter (Ficheret al., Plant Mol. Biol. 35:425-431, 1997); and the rice pistil-specificRPC312 gene promoter and its monocot homolog (JP 11098986).

According to certain embodiments of the invention, expression of aconditional lethal gene is modulated in a tissue other than areproductive tissue. For this purpose, one may choose from a number ofpromoters for genes with tissue- or cell-specific or -enhanced orinduced expression. Examples of promoters that are preferentiallyexpressed in leaves and other photosynthetically active tissues includethe chloroplast glutamine synthetase GS2 promoter from pea (Edwards etal., Proc. Natl. Acad. Sci. U.S.A. 87:3459-3463, 1990), the chloroplastfructose-1,6-biphosphatase (FBPase) promoter from wheat (Lloyd et al.,Mol. Gen. Genet. 225: 209-216, 1991), the nuclear photosynthetic ST-LS1promoter from potato (Stockhaus et al., EMBO J. 8: 2445-2451, 1989), theserine/threonine kinase (PAL) promoter and the glucoamylase (CHS)promoter from Arabidopsis thaliana. Also reported to be active inphotosynthetically active tissues are the ribulose-1,5-bisphosphatecarboxylase (RBCS) promoter from eastern larch (Larix laricina), thepromoter for the Cab gene, Cab6, from pine (Yamamoto et al., Plant CellPhysiol. 35: 773-778, 1994), the promoter for the Cab-1 gene from wheat(Fejes et al., Plant Mol. Biol. 15: 921-932, 1990), the promoter for theCab-1 gene from spinach (Lubberstedt et al., Plant Physiol.104:997-1006, 1994), the promoter for the Cab1R gene from rice (Luan etal., Plant Cell. 4:971-981, 1992), the pyruvate, orthophosphate dikinase(PPDK) promoter from Zea mays (Matsuoka et al., Proc. Natl. Acad. Sci.U.S.A. 90: 9586-9590, 1993), the promoter for the tobacco Lhcb1*2 gene(Cerdan et al., Plant Mol. Biol. 33:245-255, 1997), the Arabidopsisthaliana Suc2 sucrose-H⁺ symporter promoter (Truemit et al., Planta.196:564-570, 1995), and the promoter for the thylakoid membrane proteingenes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).Other promoters for the chlorophyll α/β-binding proteins may also beutilized in the present invention, such as the promoters for LhcB geneand PsbP gene from white mustard (Sinapis alba) (Kretsch et al., PlantMol. Biol. 28: 219-229, 1995).

For the purpose of expression of agronomic genes of interest in sinktissues other than reproductive tissue of the plant, such as the tuberof the potato plant, the fruit of tomato, or the seed of soybean,canola, cotton, Zea mays, wheat, rice, and barley, it is preferred thatthe promoters utilized in the present invention have relatively highexpression in these specific tissues. A number of promoters for geneswith tuber-specific or -enhanced expression are known, including theclass I patatin promoter (Bevan et al., EMBO J. 8:1899-1906 (1986);Jefferson. et al., Plant Mol. Biol. 14:995-1006, 1990), the promoter forthe potato tuber ADPGPP genes, both the large and small subunits, thesucrose synthase promoter (Salanoubat et al., Gene 60:47-56, 1987);Salanoubat et al., Gene 84:181-185, 1989), the promoter for the majortuber proteins including the 22 kDa protein complexes and proteinaseinhibitors (Hannapel, Plant Physiol. 101:703-704, 1993), the promoterfor the granule bound starch synthase gene (GBSS) (Visser et al., PlantMol. Biol. 17:691-699, 1991), and other class I and II patatin promoters(Koster-Topfer et al., Mol. Gen. Genet. 219:390-396 (1989); Mignery etal., Gene 62:27-44, 1988).

Other promoters can also be used to express a protein in specifictissues, such as seeds or fruits. The promoter for β-conglycinin (Chenet al., Dev. Genet. 10:112-122, 1989) or other seed-specific promoterssuch as the napin and phaseolin promoters, can be used. The zeins are agroup of storage proteins found in Zea mays endosperm. Genomic clonesfor zein genes have been isolated (Pedersen et al., Cell 29:1015-1026,1982), and the promoters from these clones, including the 15 kDa, 16kDa, 19 kDa, 22 kD, 27 kDa, and gamma genes, could also be used. Otherpromoters known to function, for example, in Zea mays include thepromoters for the following genes: waxy, Brittle, Shrunken 2, Branchingenzymes I and II, starch synthases, debranching enzymes, oleosins,glutelins, and sucrose synthases. A particularly preferred promoter forZea mays endosperm expression is the promoter for the glutelin gene fromrice, more particularly the Osgt-1 promoter (Zheng et al., Mol. Cell.Biol. 13:5829-5842, 1993). Examples of promoters suitable for expressionin wheat include those promoters for the ADPglucose pyrosynthase(ADPGPP) subunits, the granule bound and other starch synthase, thebranching and debranching enzymes, the embryogenesis-abundant proteins,the gliadins, and the glutenins. Examples of such promoters in riceinclude those promoters for the ADPGPP subunits, the granule bound andother starch synthase, the branching enzymes, the debranching enzymes,sucrose synthases, and the glutelins. A particularly preferred promoteris the promoter for rice glutelin, Osgt-1 gene. Examples of suchpromoters for barley include those for the ADPGPP subunits, the granulebound and other starch synthase, the branching enzymes, the debranchingenzymes, sucrose synthases, the hordeins, the embryo globulins, and thealeurone specific proteins.

Root specific promoters may also be used. An example of such a promoteris the promoter for the acid chitinase gene (Samac et al., Plant Mol.Biol. 25:587-596, 1994). Expression in root tissue could also beaccomplished by utilizing the root specific subdomains of the CaMV35Spromoter that have been identified (Lam et al., Proc. Natl. Acad. Sci.U.S.A. 86: 7890-7894, 1989). Other root cell specific promoters includethose reported by Conkling et al. (Plant Physiol. 93: 1203-1211, 1990).

Germination and early seedling growth promoter specificity could beprovided to drive expression of a conditional lethal transgene in agermination and early seedling growth specific or intensive process.Germination and early seedling growth promoters could be usedspecifically to affect a gene function that is essential forgermination, but its gene expression is not limited to this time in theplant growth cycle. The preferred germination specific promoter would bemost highly expressed in the appropriate tissues and cells at theappropriate developmental time to inhibit the germination enzyme or geneproduct only during germination or early seedling growth. Tissues andcells that comprise the germination and early seedling growth stages ofplants may include: the radical, hypocotyl, cotyledons, epicotyl, roottip, shoot tip, meristematic cells, seed coat, endosperm, true leaves,internodal tissue, and nodal tissue. Germination-enhanced promoters havebeen isolated from genes encoding the glyoxysomal enzymes isocitratelyase (ICL) and malate synthase (MS) from several plant species (Zhanget al, Plant Physiol. 104: 857-864, 1994); Reynolds and Smith, PlantMol. Biol. 27: 487-497, 1995); Comai et al, Plant Physiol. 98: 53-61,1992). Other promoters include SIP-seedling imbibition protein (Heck,G., Ph.D. Thesis, 1992, Washington University, St. Louis, Mo.) andothers such as a cysteine endopeptidase promoter (Yamauchi et al, PlantMol. Biol. 30: 321-329, 1996). Additionally, promoters could be isolatedfrom other genes whose mRNAs appear to accumulate specifically duringthe germination process, for example class I β-1,3-glucanase B fromtobacco (Vogeli-Lange et al., Plant J. 5: 273-278, 1994); canola cDNAsCA25, CA8, AX92 (Harada et al., Mol. Gen. Genet. 212: 466-473, 1988);Dietrich et al., J. Plant Nutr. 8: 1061-1073, 1992), lipid transferprotein (Sossountzove et al, Plant Cell 3: 923-933, 1991); or riceserine carboxypeptidases (Washio et al., Plant Phys. 105: 1275-1280,1994); and repetitive proline rich cell wall protein genes (Datta etal., Plant Mol. Biol. 14: 285-286, 1990).

Chimeric promoters that express in plants include promoters producedthrough the manipulation of known promoters to produce synthetic,chimeric, or hybrid promoters. Such promoters can also combine ciselements from one or more promoters, for example, by adding aheterologous regulatory sequence to an active promoter with its ownpartial or complete regulatory sequences (Ellis et al., EMBO J. 6:11-16,1987; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990,1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al.,Plant. Mol. Biol. 15:373-381, 1991). Chimeric promoters have also beendeveloped by adding a heterologous regulatory sequence to the 5′upstream region of an inactive, truncated promoter, i.e., a promoterthat includes only the core TATA and, optionally, the CCAAT elements(Fluhr et al., Science 232:1106-1112, 1986; Strittmatter and Chua, Proc.Nat. Acad. Sci. USA 84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet.225:65-71, 1991).

Chimeric promoters that contain regulatory elements from heterologoussources can be constructed by those skilled in the art to direct theexpression of the agronomic gene of interest and the conditional lethalgene to the desired tissue or cell in the amounts necessary to achievethe desired phenotype.

The 5′ UTR can be derived from a promoter selected to express a selectedprotein-coding region and can be specifically modified if desired so asto increase translation of mRNA. For a review of optimizing expressionof transgenes, see Koziel et al., Plant Mol. Biol. 32:393405, 1996). The5′ non-translated regions can be a native sequence obtained, forexample, from eukaryotic (e.g., plant) genes, from bacterial or viralgenes that are expressed in plant cells (e.g., genes from Agrobacteriumtumefaciens (AGRTU), or from a chimeric or synthetic gene sequence. Theoperator that is bound by an RNA-binding protein is inserted into the 5,UTR of the first DNA molecule positioned with respect to the 5′ end ofthe mRNA and the start site for protein translation such that binding ofan RNA-binding protein to the operator inhibits or substantially reducestranslation of an operably linked protein-coding sequence.

The aforesaid described genetic elements and other regulatory elementsof similar function may be substituted when appropriate by those skilledin the art of plant molecular biology to provide necessary function tothe plant expression cassettes of the present invention.

A vector may also include a screenable or scorable marker gene plantexpression cassettes. Screenable or scorable marker gene cassettes maybe used in the present invention to monitor expression in segregatingcells or progeny or loss of expression. Exemplary markers include aβ-glucuronidase or uidA gene (GUS) that encodes an enzyme for thatvarious chromogenic substrates are known (Jefferson et al., Plant Mol.Biol, Rep. 5:387-405 (1987); Jefferson et al., EMBO J. 6:3901-3907,1987); an R-locus gene, that encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues(Dellaporta et al., Stadler Symposium 11:263-282, 1988); a β-lactamasegene (Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741,1978); a gene that encodes an enzyme for that various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); aluciferase gene (Ow et al., Science 234:856-859, 1986); a xylE gene(Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105, 1983)that encodes a catechol dioxygenase that can convert chromogeniccatechols; an α-amylase gene (Ikatu et al., Bio/Technol. 8:241-242,1990); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714,1983) that encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone that in turn condenses to melanin; green flourescenceprotein (Elliot et al., Plant cell Rep. 18:707-714, 1999) and anα-galactosidase.

Agrobacterium-mediated transfer is the preferred method of the presentinvention. The use of Agrobacterium-mediated plant integrating vectorsto introduce DNA into plant cells is well known in the art. See, forexample the methods described by Fraley et al., Bio/Technology 3:629-635(1985) and Rogers et al., Methods Enzymol. 153:253-277 (1987). Theregion of DNA to be transferred is defined by the border sequences(T-DNA), and intervening DNA usually comprising a plant expressioncassette is usually inserted into the plant genome as described bySpielmann et al., Mol. Gen. Genet. 205:34 (1986).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203,1985). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes (Rogerset al., Methods Enzymol. 153:253-277, 1987). These base Agro-vectors, aswell as others known in the art can be used to incorporate the DNAconstructions and methods of the present invention. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronornically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. Back-crossing to a parental plant and out-crossing with anon-transgernic plant are contemplated, as is vegetative propagation.Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in Fehr, In: Breeding Methodsfor Cultivar Development, Wilcox J. ed., American Society of Agronomy,Madison Wis. (1987).

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011,McCabe et al, Bio/Technology 6:923 (1988), Christou et al., PlantPhysiol. 87:671-674, 1988); Brassica (U.S. Pat. No. 5,463,174); peanut(Cheng et al., Plant Cell Rep. 15:653-657 (1996), McKently et al, PlantCell Rep. 14:699-703, 1995); and pea (Grant et al., Plant Cell Rep.15:254-258, 1995), herein incorporated by reference in their entirety.

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354-5349, 1987); barley (Wan et al.,Plant Physiol 104:37-48 (1994); Zea mays (Rhodes et al., Science240:204-207 (1988), Gordon-Kamm et al., Plant Cell 2:603-618 (1990),Fromm et al., Bio/Technology 8:833-839 (1990), Koziel et al.,Bio/Technology 11: 194-200 (1993), Armstrong et al., Crop Science35:550-557 (1995); oat (Somers et al., Bio/Technology 10:1589-1594(1992); orchard grass (Horn et al., Plant Cell Rep. 7:469-472 (1988);rice (Toriyama et al., Theor Appl. Genet. 205:34-(1986), Part et al.,Plant Mol. Biol. 32:1135-1148, (1996), Abedinia et al., Aust. J. PlantPhysiol. 24:133-141 (1997), Battraw et al., Plant Mol. Biol. 15:527-538(1990), Christou et al., Bio/Technology 9:957-962 (1991); rye (De laPena et al., Nature 325:274-276 (1987); sugarcane (Bower et al., PlantJ. 2:409-416 (1992); tall fescue (Wang et al., Bio/Technology 10:691-696(1992); and wheat (Vasil et al., Bio/Technology 10:667-674 (1992); U.S.Pat. No. 5,631,152), herein incorporated by reference in their entirety.

Assays for gene expression based on the transient expression of clonednucleic acid vectors have been developed by introducing the nucleic acidmolecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522, 1990). Transientexpression systems may be used to functionally dissect gene constructs(see generally, Mailga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press, 1995). It is understood that any of the nucleicacid molecules of the present invention can be introduced into a plantcell in a permanent or transient manner in combination with othergenetic elements such as promoters, leaders, transit peptide sequences,enhancers, introns, 3′ nontranslated regions and other elements known tothose skilled in the art that are useful for control of transgeneexpression in plants.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials that describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Mailga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Clark et al., Plant Molecular Biology: ALaboratory Manual, Springer, New York (1997); and Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, (1990).

Examples of suitable structural genes of agronomic interest envisionedby the present invention would include but are not limited to one ormore genes for insect tolerance, such as a Bacillus thuringiensis (B.t.)gene, disease tolerance such as genes for fungal disease control, virusdisease control, bacteria disease control and nematode disease control;herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 9931248 herein incorporated by reference in itsentirety, U.S. Pat. No. 5,689,052 herein incorporated by reference inits entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in its entirety. The herbicide tolerancepolynucleotide sequences include, but are not limited to polynucleotidesequences encoding for proteins involved in herbicide tolerance encodingfor 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, described inU.S. Pat. Nos. 5,627,061, and 5,633,435, herein incorporated byreference in its entirety; Padgette et al. (1996) Herbicide ResistantCrops, Lewis Publishers, 53-85, and in Penaloza-Vazquez, et al. (1995)Plant Cell Reports 14:482-487) and aroA (U.S. Pat. No. 5,094,945) forglyphosate tolerance; bromoxynil nitrilase (Bxn) for Bromoxyniltolerance (U.S. Pat. No. 4,810,648); phytoene desaturase (crtl (Misawaet al, (1993) Plant Journal 4:833-840, and (1994) Plant Jour 6:481-489)for tolerance to norflurazon, acetohydroxyacid synthase (AHAS,Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193); and the bargene for tolerance to glufosinate (DeBlock, et al. (1987) EMBO J.6:2513-2519. Herbicides for which transgenic plant tolerance has beendemonstrated and the method of the present invention can be appliedinclude, but are not limited to: glyphosate, glufosinate, sulfonylureas,imidazolinones, bromoxynil, delapon, cyclohezanedione,protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides.

DNA Constructs

DNA constructs in an aspect of the present invention contain at leasttwo T-DNAs and the DNA segments residing between the two T-DNAs. EachT-DNA comprises an Agrobacterium right border (RB) and left border (LB)sequence, and contain 1 or more plant expression cassettes. It is commonto construct a T-DNA containing multiple plant expression cassettes.FIG. 1, pMON42061 illustrates a single T-DNA construct containing 3plant expression cassettes, (P-FMV.34S/At.CTP2/aroA:CP4/E93′;PCaMV.35S/nptII/Nos 3′; and P-CaMV.35S:en/GUS:1/intron/GUS:1/Nos 3′) andFIG. 8, pMON42073 illustrates a single T-DNA construct containing 4plant expression cassettes (P-Os.Act1/GUS:1/intron/GUS:1/T-TaHsp17;P-CaMV.35S/nptII/Nos3′; P-CaMV.35S:en/I-Os.Act1/L-Ta.Cab/GFP/T-Ta.Hsp17,P-Os.Act1/I-OsAct1/At.CTP2/aroA:CP4/Nos 3′ between a RB and a LB. Asillustrated in pMON42061 and pMON42073 multiple expression cassettescontaining agronomic genes of interest, selectable and scorable markergenes can be included in a single T-DNA. These constructs also haveunique endonuclease restriction sites for the further engineering of theconstruct, for example, the FseI site of pMON42061 and the EcoO109 siteof pMON42073. Additional endonuclease restriction sites that can be usedfor further engineering of the DNA constructs of the present inventioninclude, but are not limited to, NotI, Pmel, XhoI, SmaI, HindIII, andNheI. Those of ordinary skill in the art of plant molecular biology canremove, replace, modify or add restriction sites and polylinker DNAmolecules to provide modification of the DNA constructs that enhance themobility of DNA segments, genetic elements, expression cassettes, andT-DNAs into and out of the DNA constructs.

A DNA construct of this aspect of the present invention contains atleast two T-DNAs allowing for a multitude of combinations of plantexpression cassettes. Also, the DNA segments between the T-DNAs can beused to insert various plant and bacterial expression cassettes. The DNAconstructs that comprise the Two T-DNAs can include variousmodifications that decrease T-DNA insert complexity and increaseindependent segregation. These include the length of the DNA segmentsresiding between the T-DNAs in a DNA construct. This length is avariable that can be manipulated to enhance the separate integration ofthe T-DNAs in the plant genome and low copy number of the insertions.The range of the length of the DNA segments can be from about 1000nucleotide base pairs (1 kb) to about 15 kb. The DNA construct,pMON51652, illustrated in FIG. 2 is an example of a construct with asmall DNA segment spacer of about 1400 base pairs and a larger segmentof about 4.5 kb. The DNA construct, pMON51661, illustrated in FIG. 6 isan example of a construct with DNA segment spacers of about 4 kb. Bothof these constructs contain a unique NotI site for insertion ofadditional plant expression cassettes of agronomic genes of interest anda unique Fse1 site for insertion of a conditional lethal gene expressioncassette.

Another modification is the orientation of the border sequences of theT-DNAs, this can also affect the efficiency of independent segregationof the T-DNAs. In pMON51652, the clockwise orientation of the T-DNAs isRB-LB for T-DNA 1 that contains the plant expression cassette expressingthe aroA:CP4 gene (glyphosate tolerance), and RB-LB for T-DNA 2 thatcontains the plant expression cassettes expressing nptII (kanamycintolerance) and GUS: 1 (β-glucuronidase, visual marker). The pMON51652construct has a unique FseI and SacII endonuclease enzyme site in theDNA segment that can be used to insert plant expression cassettes thatprovide conditional lethal gene products to further enhance independentsegregation of the T-DNAs. The DNA construct, pMON42071, illustrated inFIG. 10, is similar in design to pMON51652 except that it contains inits T-DNAs genetic elements designed for enhanced expression in monocotplants. The clockwise orientation of the borders of the T-DNAs in thisconstruct is RB-LB for T-DNA 1 (GUS; nptII) and RB-LB for T-DNA 2 (GFP;aroA:CP4). The DNA construct, pMON51653, illustrated in FIG. 3, has aclockwise orientation of the border sequences of RB-LB for T-DNA 1(aroA:CP4) and LB-RB for T-DNA 2 (GUS; nptII). The pMON42072 construct,illustrated in FIG. 11, is similar in design to pMON51653 except that itcontains in its T-DNAs genetic elements designed for enhanced expressionin monocot plants, its T-DNAs have a clockwise orientation of the bordersequences of RB-LB for T-DNA 1 (GUS; nptII) and LB-RB for T-DNA 2(aroA:CP4; GFP). The constructs has a unique endonuclease enzyme sitesin the DNA segment that can be used to insert plant expression cassettesthat provide the conditional lethal gene products to further enhanceindependent segregation of the T-DNAs. Constructs that have two T-DNAsorientated RB-first T-DNA-LB, LB-second T-DNA-RB can show an increase inindependent segregation of the T-DNAs, reduced occurrence of theconstruct DNA segments and an increase in detection of expression of thetransgenes from each of the T-DNAs.

The DNA constructs of the present invention are transformed into plantcells by an Agrobacterium mediated transformation methods. Variousstrains of Agrobacterium are hosts for the DNA constructs and functionto transfer the T-DNAs of the constructs dependent on the presence ofcompatible transfer elements. The element trfA (pRK2TRFA) providescomplementation of the transfer function for strain LBA4404 (an octopinestrain) and when inserted into pMON51653 to create pMON54237 (FIG. 4)will permit the T-DNAs of this construct to be transferred into plantsby LBA4404. The construct, pMON54237 contains a unique NotI site forinsertion of additional plant expression cassettes of agronomic genes ofinterest and a unique Fse1 site for insertion of a conditional lethalgene expression cassette. For comparison, pMON51653 and pMON51652 aretransferred into plants via strain ABI (a nopaline strain).Agrobacterium strain differences is a modification of the T-DNA transfermethod that can influence the efficiency of T-DNA transfer and thesegregation of the T-DNAs in the transformed plant. The border regionsare derived from octopine or nopaline strains of Agrobacterium. Theconstruct, pMON51676 (FIG. 7) contains two T-DNAs, the T-DNA 1 containsa RB nop and a LB nop region from a nopaline strain, and T-DNA 2contains a RB oct and a LB oct region from an octopine strain. Thesearrangement of T-DNA borders in combination with homologous andheterologous Agrobacterium nopaline and octopine strains will allow formore control of the T-DNA processing and provides a differential thatcan be manipulated for the preferential insertion of a T-DNA. Thedifferent strains of disarmed Agrobacterium that serve as the host forthe DNA constructs, i.e., nopaline and octopine strains when combinedwith T-DNAs with that vary in the origin of the border sequences canprovide enhancement to insertion of the T-DNAs when these strains aremixed together in a co-transformation Agrobacterium mediated planttransformation method. In this aspect of the invention, the DNAconstructs that are used in a co-transformation method with differentstrains may contain only one T-DNA and a conditional lethal generesiding in the DNA segment. Additionally, DNA constructs can beselected for use in a co-transformation method with different strains,wherein at least one of the DNA constructs does not contain aconditional lethal gene.

The present invention also provides modification in a DNA construct toenhance the integrity of the T-DNAs and reduce the often observed leftborder readthrough. The Agrobacterium virulence genes, virD1 and virD2encode for endonucleases that nick within the 25 bp RB and LB nucleotidesequences, providing a template with clearly defined 5′ and 3′ ends. Theinclusion of the virD1 and virD2 genes in the DNA segments outside ofthe T-DNAs will increase efficiency of nicking at the borders,particularly the LB, and prevent border readthrough during plant genomicintegration. The prevention of border readthrough will increase theintegrity of the T-DNA and enhance the segregation of the T-DNAs in theplant genome and therefore the quality of transformed events. Theexpression of virD1 and virD2 genes is by the native virD promoter whichhas been PCR amplified and placed upstream of the coding regions. Theconstruct, pMON51658 (FIG. 5) contains the virD1 and virD2 expressioncassettes in the DNA segment located between T-DNA 1 (aroA:CP4) andT-DNA 2 (nptII; GUS) of this construct. Additional unique endonucleasesites, NdeI and FseI are positioned to permit the insertion ofconditional lethal gene expression cassettes. pMON42070 (FIG. 9)contains within its T-DNAs, plant expression cassettes that containgenetic elements for enhanced expression in monocot plants, the virD1and virD2 genes, and a unique endonuclease site EcoO109 for insertion ofconditional lethal gene expression cassettes. The constructs containingthe virD1 and virD2 genes and the conditional lethal genes will have theproperties of enhanced termination at the border sequences and a methodto eliminate any plants that have the T-DNAs linked at the same locus.Additional plant expression cassettes containing agronomic genes ofinterest can be inserted into the construct at the endonuclease sitesresiding within the T-DNAs as illustrated in FIGS. 5 and 9.Agrobacterium mediated transformation of dicot and monocot plant cellswith genetic elements designed for enhanced expression in these plantsare known to those skilled in the art of plant molecular biology. Thesegenetic elements in operable linkage contained within the two T-DNAs ofa DNA construct and containing a conditional lethal gene is within thescope of the present invention. In subsequent generations of the plantstransformed with these DNA constructs, the plants are treated with aprotoxin that is processed by the conditional lethal gene product into aphytotoxin. Surviving plants contain T-DNAs that can be geneticallyseparated in breeding populations.

Conditional Lethal Gene Elements

The conditional lethal gene, pehA, expression cassette is illustrated inpMON9443 construct (FIG. 12). Expression cassettes and regulatoryelements found in the DNA segment outside of the plant expressionelements contained in the T-DNA are common in many plasmid DNA backbonesand function as plasmid maintenance elements, these include, but are notlimited to, the aad (Spc/Str) gene for bacterialspectinomycin/streptomycin resistance, the pBR322 ori (ori322) thatprovides the origin of replication for maintenance in E. coli, the bomsite for the conjugational transfer into the Agrobacterium tumefacienscells, and a DNA segment is the 0.75 kb oriV containing the origin ofreplication from the RK2 plasmid.

Within the T-DNA borders are various plant expression cassettes. One ofthese contains the enhanced CaMV 35S promoter (P-CaMV.35S.en) (U.S. Pat.No. 5,359,142, herein incorporated in its entirety) in front of apoly-cloning site where the conditional lethal gene pehA:2 (U.S. Pat.No. 5,254,801) is encoded, followed by the pearibulose-1,5-bis-phosphate carboxylase small subunit E9 3′non-translated region (Coruzzi et al., EMBO J. 3:1671, 1984). A scorablemarker gene, GUS:1 (U.S. Pat. No. 5,268,463, herein incorporated byreference in its entirety) expression is driven by the enhanced CaMV35Spromoter and terminated by the 3′-nontranslated region of the nopalinesynthase gene (NOS 3′). A chimeric kanamycin resistance gene thatpermits selection of transformed plant cells, P-CaMV.35S/nptII/NOS 3′consists of the cauliflower mosaic virus (CaMV) 35S promoter (U.S. Pat.No. 5,858,742, herein incorporated in its entirety), the neomycinphosphotransferase type II (nptII) gene (U.S. Pat. No. 6,174,724, hereinincorporated in its entirety) and the 3′-nontranslated region of thenopaline synthase gene (NOS 3′). The expression cassette containing thepehA:2 gene is mobilized from pMON9443 to comprise a functional locationin anyone of the DNA constructs of the present invention and provideconstitutive expression of the conditional lethal gene product therefrom.

Constitutive expression of the pehA:1 (SEQ ID NO:1) and pehA:2 (SEQ IDNO:2) phosphonate monoester hydrolase enzyme in plants has no directeffect on plant growth or development. Only in the present of aphosphonate ester of glyphosate protoxin does the enzyme function toconvert the protoxin into a toxin in the tissue in which it isexpressed. The pehA gene constitutive expression cassette, for example,the P-CaMV.35S:en/pehA:2/E9 3′ of pMON9443 can be inserted into the twoT-DNA constructs of the present invention at the unique endonucleasesites residing in the DNA segments outside of the two T-DNAs. Theprogeny of parents transformed with the DNA constructs of the presentinvention containing a constitutively expressed PEHA enzyme will bekilled when treated with a phosphonate ester of glyphosate. Theremaining progeny can be screened by bioassay, molecular diagnostics,field performance, trait efficacy or other methods particular to theagronornic genes of interest and known to those skilled in the art.

Tissue specific expression conditional lethal genes in combination withthe DNA constructs of the present invention are particularly useful foreliminating unwanted cell or tissue types during breeding or propagationof the progeny. The DNA construct, pMON9430, illustrated in FIG. 13,contains a plant expression cassette where the peha: 1 gene expressionis driven by a tissue specific promoter. In this example, the tissuespecific promoter P-127a from tomato (U.S. Pat. No. 5,254,801) expressesin tapetal cells, a male tissue. Other promoters that have specific celltype expression patterns can be used to drive the expression of the pehagene coding sequences. These promoters may be particularly selected toexpress in dicot or monocot plants, more preferably selected to expressin the gametophytic cell of a transgenic dicot or monocot plant, or morepreferably selected to express in the pollen cells of a transgenic dicotor monocot plant. A DNA molecule containing the peha: expressioncassette from pMON9430 can be mobilized by digestion with NotI orHpaI/SmaI restriction endonucleases and inserted into the two T-DNAconstructs of the present invention at the unique endonucleaserestriction sites illustrated in the FIGS. 3-11. These insertions canoccur in the DNA segments outside of the T-DNAs to select against plantscontaining these segments or the insertions can occur inside of theT-DNAs to select against plant cells or plants that contain a particularT-DNA.

The argE gene (N-acetyl-L-ornithine deacetylase) and its substrateN-acetyl-L-phosphinothricin (Kriete et al. Plant J. 9:809-818, 1996;Beriault et al. Plant Physiol. 121:619-628, 1999; herein incorporated byreference in its entirety) is another example of a protoxin substratethat can be converted into a toxic molecule in plants. An argE geneplant expression cassette can be inserted into the two T-DNA constructsof the present invention at sites in the DNA constructs located in theDNA segments between the two T-DNAs. Alternatively, the argE gene plantexpression cassette can be inserted into one of the T-DNAs and used toselect against the T-DNA at some future stage of plant development or inthe process of plant breeding or plant cultivation. Tissue specificexpression can direct the expression of the enzyme and hence theconversion of protoxin to certain cells or at a certain stage ofdevelopment depending on the need. For example, the TA29 promoterdriving expression of the argE coding sequence in tapetal cell was ableto specifically able to convert the protoxin substrate into a toxin inthese cells resulting in male sterility (Kriete et al. Plant J.9:809-818, 1996, the entirety of which is herein incorporated byreference).

Glyphosate metabolism (degradation) has been examined in a wide varietyof plants and little degradation has been reported in most of thosestudies. In those instances where degradation has been reported, theinitial breakdown product is usually aminomethylphosphonate (AMPA)(Coupland, 1985. The Herbicide Glyphosate pp 25-34, Butterworths publ.;Marshall et al., 1987. Pestic. Sci 18:65-77). AMPA has been reported tobe much less phytotoxic than glyphosate for most plant species (Franz,1985. The Herbicide Glyphosate pp 25-34, Butterworths publ.) but not forall plant species (Tanaka et al., 1988. J. Fac. Agr. Kyushu Univ.30:209-223). A number of pure cultures of bacteria have been identifiedthat degrade glyphosate by one of the two known routes (Moore et al.,1983. Appl. Environ. Microbiol. 46:316-320; Talbot et al., 1984. CurrentMicrobiol. 10:255-260; Shinabarger and Braymer, 1986. J. Bacteriol.168:702-707; Balthazor and Hallas, 1986. Appl. Environ. Microbiol.51:432-434; Kishore and Jacob, 1987. J. Biol. Chem. 262:12164-12168;Wackett et al., 1987. J. Bacteriol. 169:710-717; Pipke et al., 1987.Appl. Environ. Microbiol. 53:974-978; Pipke et al., 1987. Appl. Environ.Microbiol. 54:1293-1296; Hallas et al., 1988. J. Industrial Microbiol.3:377-385; Jacob et al., 1988. Appl. Environ. Microbiol. 54:2953-2958;Liu et al., 1991. Appl. Environ. Microbiol. 57:1799-1804). Pure culturescapable of degrading glyphosate to AMPA have been reported for aFlavobacterium sp. (Balthazor and Hallas, 1986), for a Pseudomonas sp.(Jacob et al., 1988. Appl. Environ. Microbiol. 54:2953-2958) and forArthrobacter atrocyaneus (Pipke and Amrhein, 1988). In addition, a largenumber of isolates that convert glyphosate to AMPA have been identifiedfrom industrial activated sludges that treat glyphosate wastes (Hallaset al., 1988).

Glyphosate oxidoreductase (GOX) (SEQ ID NO:3) degrades glyphosate toAMPA. This enzyme has been engineered into plants with DNA constructs,for example, pMON17164, illustrated in FIG. 14, as well as otherconstructs described in U.S. Pat. No. 5,463,175, the entirety of whichis herein incorporated by reference, and confers glyphosate tolerance tothese plants. AMPA being generally less phytotoxic than glyphosate isvegetatively tolerated by the transgenic plants. However, plant malegametophytic cells are particularly sensitive to AMPA. AMPA alone canfunction as a gametocide in plants. A transgenic plant that isexpressing a glyphosate resistant EPSPS, for example, aroA:CP4, althoughhighly tolerant to glyphosate still retains sensitivity to AMPA. The GOXgene expression directed by a pollen specific promoter convertsglyphosate to AMPA in a glyphosate tolerant plant. The increased levelsof AMPA would be toxic to the pollen cells expressing GOX and would beimpaired or killed. In the present invention, a GOX plant expressioncassette functions as a conditional lethal gene in a glyphosate tolerantplant. The GOX expression cassette can be inserted in the DNA segmentsbetween two T-DNAs and transgenic plants produced. When the plants aretreated with glyphosate, the pollen cells containing the GOX expressioncassette will be impaired or killed, leaving only the pollen thatcontains the segregated T-DNAs. In a similar method, when the GOXexpression cassette is included within a T-DNA, then the pollencontaining that T-DNA will be specifically impaired or killed. Thisapplication has utility for limiting the outcrossing of transgenes in aplant breeding environment and in a field environment by treatment withglyphosate.

Additional conditional lethal genes include, but are not limited to theiaaH gene encoding indoleacetamide hydrolase which can convert non-toxiclevels of naphthalene acetamide into toxic levels of the auxin,naphthalene acetic acid (Klee et al., Genes Dev. 1:86-96, 1987, theentirety of which is herein incorporated by reference), the bacterialcytosine deaminase gene has been shown to function as a conditionallethal gene for negative selection in plants (Kobayashi et al. Jpn. J.Genet. 70:409-422; Perera et al. Plant Mol. Biol. 23:793-799, theentirety of which is herein incorporated by reference), and the viralthymidine kinase (Czako et al. Plant Physiol 104:1067-1071, 1994, theentirety of which is herein incorporated by reference).

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense. All references cited herein are hereby expressly incorporatedherein by reference.

EXAMPLES Example 1

The use of conditional lethal transgenes in two T-DNA constructsinvolves the construction of plant transformation vectors that includethe appropriate combination of genetic elements necessary to directadequate expression levels in target tissues. For monocotyledonous cropplants, a monocot vector that utilizes a plant expression cassette thatcontains a promoter (P) and first intron (1) for example, from the rice(Os) actin gene (P-Os.Act1/I-Os.Act1 (U.S. Pat. No. 5,641,876, hereinincorporated by reference in its entirety). DNA molecules that encodetargeting sequences may be used when determined by those skilled in theart that it would contribute to the desired phenotype, for example theplastid transit peptide sequence from the Arabidopsis thaliana (At) EPSPsynthase gene (At.CTP2) (Klee et al. Mol. Gen. Genet. 210:437-442, theentirety of which is herein incorporated by reference) would be anappropriate choice. The choice of the conditional lethal transgenecoding sequence is dependent on the method most useful for the operator.For example, constitutive expression of the coding sequences encodingphosphonate monoester hydrolase, carboxylate monester hydrolase,N-acetyl-L-ornithine deacetylase, and P450 monooxygenase CPY105A1 intransgenic plants will result in impairment or death of the plant afterapplication of the respective protoxin. Modification of the nucleotideDNA sequence for each conditional lethal gene coding sequence forenhanced expression in monocots can be determined by those skilled inthe art. A plant expression cassettes needs apolyadenylation/termination (T) region, such a region isolated from theAgrobacterium tumefaciens nopaline synthase gene (T-AGRTU.nos), would bean appropriate choice.

For specific dicotyledonous species, a plant expression vector thatutilizes the promoter and 5′ untranslated region (including intron I),for example, the plant elongation factor 1a gene (Elfα-A1) as describedin U.S. Pat. No. 5,177,011, herein incorporated by reference in itsentirety, constitutive plant viral promoters of the caulimovirus family,including by not limited to Cauliflower mosaic virus (P-CaMV.35S, U.S.Pat. No. 5,858,742, herein incorporated by reference in its entirety),and Figwort mosaic virus (P-FMV.34S, U.S. Pat. No. 5,378,619, hereinincorporated by reference in its entirety). Transit peptide sequencescan be used when necessary to enhance the desired phenotype, forexample, the chloroplast transit peptide from the Arabidopsis thalianaEPSP synthase gene (At.CTP2). Termination regions for example, thepolyadenylation/3′ termination region from the Pisum sativumribulose-1,5-bisphosphate carboxylase gene (E9 3′) is a useful geneticelement of a plant expression cassette.

Example 2

Transgenic corn plants can be produced by an Agrobacterium mediatedtransformation method. The constructs, pMON42070 (FIG. 9), pMON42071(FIG. 10), pMON42072 (FIG. 11), a 1 T-DNA construct pMON42073 (FIG. 8)as a control construct, and constructs derived from these containing aconditional lethal gene inserted into the DNA segment located betweenthe two T-DNAs are used to transform corn cells. For example, pMON73105(FIG. 15, [RB]P-CaMV.35S/nptII/nos3′[LB];[RB]P-Os.Act1/GUS:1/T-Ta.Hsp17[LB]; P-FMV/I-Os.RbcS/pehA2/nos3′; andpMON73107 (FIG. 16, [RB]P-CaMV.35S/nptII/nos 3′[LB]; P-FMV/pehA2/nos 3′;[RB] P-Os.Act1/GUS:1/E9 3′[LB]) construct of the present invention hasan expression cassette for pehA2 inserted in one of the DNA segments ofthe construct. DNA constructs of the present invention have aconditional lethal gene inserted into both DNA segments, for example,pMON73106 (FIG. 17, [RB]P-CaMV.35S/nptII/nos3′[LB];P-ScBV/I-Os.Act1/pehA2/nos3′; [RB]P-Os.Act1/GUS;1/T-Ta.Hsp 17-[LB];P-FMV/I-Os.RbcS/pehA2/nos3′). These DNA constructs have a conditionallethal gene inserted into at least one T-DNA for use in eliminating thisT-DNA at a desired stage in the breeding or propagation of thetransgenic plant, for example pMON73108 (FIG. 18,[RB]P-FMV/I-Os.RbcS/pehA2/nos3′; P-CaMV.35S/nptII/nos 3′[LB];[RB]P-Os.Act1/GUS:1/T-Ta.Hsp17[LB].

The construct is transferred into Agrobacterium by a triparental matingmethod (Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351). Liquidcultures of Agrobacterium are initiated from glycerol stocks or from afreshly streaked plate and grown overnight at 26° C.-28° C. with shaking(approximately 150 rpm) to mid-log growth phase in liquid LB medium, pH7.0 containing 50 mg/l kanamycin, 50 mg/l streptomycin and spectinomycinand 25 mg/l chloramphenicol with 200 μM acetosyringone (AS). TheAgrobacterium cells are resuspended in the inoculation medium (liquidCM4C) and the density is adjusted to OD₆₆₀ of 1. Freshly isolated TypeII immature HiIIxLH198 and Hill corn embryos are inoculated withAgrobacterium containing the construct and co-cultured 2-3 days in thedark at 23° C. The embryos are then transferred to delay media (N61-100-12/micro/Carb 500/20 μM AgNO3) and incubated at 28° C. for 4 to 5days. All subsequent cultures are kept at this temperature. Coleoptilesare removed one week after inoculation. For constructs that containglyphosate tolerance genes, the corn embryos are transferred to thefirst selection medium (N61-0-12/Carb 500/0.5 mM glyphosate). Two weekslater, surviving tissue are transferred to the second selection medium(N61-0-12/Carb 500/1.0 mM glyphosate). Subculture surviving callus every2 weeks until events can be identified. This will take 3 subcultures on1.0 mM glyphosate. Once events are identified, bulk up the tissue toregenerate. For constructs that contain nptII tolerance genes, the cornembryos are transferred to the first selection medium (N61-0-12/Carb500/50 mg/L paramomycin). Two weeks later, surviving tissue aretransferred to the second selection medium (N61-0-12/Carb 500/100 mg/Lparamomycin). Subculture surviving callus every 2 weeks until events canbe identified. This will take 3 subcultures on 100-200 mg/L paramomycin.Once events are identified, bulk up the tissue to regenerate.

For regeneration, callus tissues are transferred to the regenerationmedium (MSOD 0.1 μM ABA) and incubated for two weeks. The regeneratingcalli are transferred to a high sucrose medium and incubated for twoweeks. The plantlets are transferred to MSOD media in culture vessel andkept for two weeks. Then the plants with roots are transferred intosoil.

The corn plants are grown in a greenhouse to maturity, selfed and seedscollected. The seeds are planted in pots in conditions that permitgermination and seedling growth. The plants are treated with aneffective dose of the protoxin that corresponds to the conditionallethal gene. Leaf tissue samples are collected from the undamaged orsurviving seedlings and assayed for the GUS activity by MOPS method (seefor example, Jefferson et al., EMBO J. 6:3901, 1987, the entirety ofwhich is herein incorporated by reference). The seedlings are alsoassayed for the presence of the EPSPS:CP4 enzyme by ELISA (Padgette etal. Crop Science 35:1451-1461, 1995). The segregation ratio isdetermined for the two T-DNAs. Plants with only the T-DNA containing theagronomic genes of interest are selfed, backcrossed or outcrossed inaccordance with standard breeding methods (Fehr, Principles of CultivarDevelopment Vol. 1, pp. 2-3 (1987), the entirety of which is hereinincorporated by reference.).

Transgenic wheat plants are produced by transforming immature embryos ofwheat (Triticum aestivum L) cultivar Bobwhite with the constructs of thepresent invention. The constructs, pMON42070 (FIG. 9), pMON42071 (FIG.10), pMON42072 (FIG. 11), a 1 T-DNA construct pMON42073 (FIG. 8) as acontrol construct, and constructs derived from these containing aconditional lethal gene inserted into the DNA segment located betweenthe two T-DNAs are used to transform corn cells. A construct of thepresent invention is transferred into Agrobacterium by a triparentalmating method (Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351). Wheatembryos are isolated from the immature caryopsis 13-15 days afterpollination, and cultured on CM4C (Table 1.) for 3-4 days. The embryosshowing active cell division, but no apparent callus formation areselected for Agrobacterium infection. TABLE 1 Supplemental Components inBasal Media¹ Components CM4 CM4C MMS.2C MMS0 2,4-D (mg/l) 0.5 0.5 0.2 —Pichloram (mg/l)² 2.2 2.2 Maltose (g/l) 40.0 40.0 40.0 40.0 Glutamine(g/l) 0.5 0.5 Magnesium Chloride (g/l) 0.75 0.7 Casein Hydrolysate (g/l)0.1 0.1 MES (g/l) 1.95 1.95 1.95 Ascorbic Acid (mg/l)² 100.0 100.0 100.0Gelling Agent (g/l)³ 2(P) 2(P) 2(G) 2(G)¹All media contain basal salts (MS basal salts) and vitamins (MSvitamins) from Murashige and Skoog (1962). The pH in each medium isadjusted to 5.8.²Filter-sterilized and added to the medium after autoclaving.³Phytagel ™ (P) or Gelrite ®(G).

Liquid cultures of Agrobacterium containing the constructs of thepresent invention are initiated from glycerol stocks or from a freshlystreaked plate and grown overnight at 26° C.-28° C. with shaking(approximately 150 rpm) to mid-log phase (OD₆₆₀=1-1.5) in liquid LBmedium, pH 7.0 containing 50 mg/l kanamycin, 50 mg/l streptomycin andspectinomycin and 25 mg/l chloramphenicol with 200 μM acetosyringone(AS). The Agrobacterium cells are resuspended in the inoculation medium(liquid CM4C) and the density is adjusted to OD₆₆₀ of 1. The immatureembryos cultured in CM4C medium are transferred into sterile petriplates (16×20 mm) or wells of a 6-well cell culture plate (CostarCorporation, Cambridge, Mass.) containing 10 ml of inoculation mediumper petri plate or 5 ml per cell culture cluster plate. An equal amountof the Agrobacterium cell suspension is added such that the finalconcentration of Agrobacterium cells is an OD₆₀₀ of 0.5. In mostexperiments, pluronic F68 is added to the inoculation mixture at a finalconcentration of 0.01%. The ratio between the Agrobacterium and immatureembryos is about 10 ml: 20-200 IEs. The inoculation is allowed toproceed at 23° C.-26° C. from 5-60 minutes. After the inoculationperiod, the remaining Agrobacterium cells are removed from the explantsby using vacuum aspiration equipment. A piece of sterile Whatman No. 1filter paper (to fit the size of the petri plate) is placed in each of60×15 or 60×20 mm petri dishes. Two hundred μl of sterile water isplaced in the middle of the filter paper. After 2-3 minutes, theinoculated immature embryos are placed in the plates. Usually, 20-50explants are grouped as one stack (about 1 cm in size and 60-80mg/stack), with 4-5 stacks on each plate. The plates are immediatelycovered with Parafilm® and then co-cultivated in the dark at 24° C.-26°C. for 2-3 days. The co-cultivated PCIEs are transferred CM4C+500 mg/lcarbenicillin medium (delay medium) at dark. After 7 days on the delaymedium, the immature embryos are transferred to CM4C supplemented with 2mM glyphosate and 500 mg/l carbenicillin for selection for one week.Then calli are transferred to MMS0.2C+0.1 mM glyphosate +250 mg/lcarbenicillin medium for 2 weeks under light for further selection.Embryogenic calli are transferred to a second regeneration medium MMSOCwith lower glyphosate concentration (0.02 mM) and 500 mg/L carbenicillinfor plant regeneration. Those embryogenic calli are transferred ontofresh medium every two weeks. Regenerated plantlets are transferred toSundae cups (Sweetheart Cup Company, Chicago, Ill.) containing thesecond regeneration medium for further growth and selection. When rootsare well established from transgenic plants the plants are transferredto soil for further evaluation.

The wheat plants are grown in a greenhouse to maturity, selfed and seedscollected. The seeds are planted in pots in conditions that permitgermination and seedling growth. The plants are treated with aneffective dose of the protoxin that corresponds to the conditionallethal gene. Leaf tissue samples are collected from the undamaged orsurviving seedlings and assayed for the reporter gene, for example, GUSby assays performed by routine methods (see for example, Jefferson etal., EMBO J. 6:3901, 1987). The seedlings are also assayed for thepresence of the CP4 EPSPS enzyme or NPTII by ELISA depending on theconstruct used. The segregation ratio is determined for the two T-DNAs.Plants with only the T-DNA containing the agronomic genes of interestare selfed, backcrossed or outcrossed in accordance with standardbreeding methods.

Transgenic Arabidopsis plants are produced with the DNA constructs ofthe present invention using the method of vacuum infiltration ofAgrobacterium containing the DNA constructs (Bechtold et al., C R AcadParis Life Sci 316: 1194-1199, the entirety of which is hereinincorporated by reference). The constructs, a 1 T-DNA constructpMON42061 (FIG. 1) as a control construct, pMON51652 (FIG. 2), pMON51653(FIG. 3), pMON54237 (FIG. 4), pMON51658 (FIG. 5), pMON51661 (FIG. 6),pMON51676 (FIG. 7) and constructs derived from these containing aconditional lethal gene inserted into the DNA segment located betweenthe two T-DNAs are used to transform Arabidopsis cells. Seeds are pottedin soil in trays in a growth chamber adjusted for 24° C., 16 hour light(120 μE m⁻² s⁻¹) cycle to permit normal growth and development of theseedling plants. The Arabidopsis seedling plants are treated with aneffective dose of the protoxin that corresponds to the conditionallethal gene capability to convert the protoxin into a toxin. Leaf tissuesamples are collected from the undamaged or surviving seedlings andassayed for the GUS activity. The seedlings are also assayed for thepresence of the CP4 EPSPS enzyme or NPTII by ELISA depending on theconstruct used. The segregation ratio is determined for the two T-DNAs.Plants with only the T-DNA containing the agronomic genes of interestare selfed, backcrossed or outcrossed in accordance with standardbreeding methods (Fehr, Principles of Cultivar Development Vol. 1, pp.2-3 (1987), the entirety of which is herein incorporated by reference).

Transgenic soybean (Glycine max) containing DNA constructs of thepresent invention expressing a glyphosate tolerance gene are generatedby Agrobacterium tumefaciens-mediated transformation of cotyledonexplants. Asgrow (Stuttgart, Ak.) soybean cultivars A3244, A4922, andA5547 soybean seeds are soaked in sterile distilled water (SDW) at roomtemperature for either two hours or three minutes, drained, and leftmoist for two hours. Bean Germination Media (BGM, Table 2) is addedafter two hours to 2-3 times the depth of the seed volume and incubatedat room temperature in the dark for nine to eleven hours.

At eleven to thirteen hours from initiation of germination, the seedaxes are removed from seeds and held in sterile distilled water.Explants are rinsed two to three times with sterile distilled water,drained and divided into sets of 50-300. Each set is placed into avessel along with either ABI or LBA4404 strains of Agrobacteriacontaining the DNA construct, induced with 0.2 mM Acetosryingone, 1.0 μMgalacturonic acid and 0.25 mg/L GA₃ Examples of vessels included a 25milliliters (ml) glass test tube with two ml of Agrobacterium, a 125 mlglass flask with 5-10 ml Agrobacterium, or a Plantcon™ with 20-50 mlAgrobacterium.

Each vessel is held in a sonicator (L&R Ultrasonics Model PC5 or QS140,)with 500-2000 milliliters of sterile distilled water plus 0.1% TritonX100 and sonicated for five to sixty seconds. In some cases, freshinoculum is added following sonication. Explants are inoculated forabout one hour while shaking on an orbital shaker at approximately 90RPM.

Explants are then co-cultured on one piece of Whatman grade 1 filterpaper with one to seven mil of 1/10× Gamborg's B5 media (Gamborg et al.,Exp. Cell Res., 50:151-158, 1968) containing 1.68 mg/L BAP, 3.9 g/L MESwith inducers as listed above for two to four days at approximately 23°C., dark.

After co-culture, explants are transferred to Woody Plant Media (WPM)(McCown et al., Proc. International Plant Propagation Soc., 30:421,1981) containing 2% sucrose, 60 mg/L Benomyl, 75 μM glyphosate pluscombinations of the following antibiotics; 200 mg/L Carbenecillin,100-200 mg/L Cefotaxime, 100 mg/L Timetin. Cultures are incubated atapproximately 28° C. with a 16 hour light, 8 hour dark photoperiod. Atransfer to fresh media is made after seven to fourteen days. In somecases, a third transfer is made after an additional ten to fourteendays.

Shoots are harvested between three and seven weeks post-inoculation.Shoots rooted on BRM (Table 2) with 25 to 40 μM glyphosate. TABLE 2 BEANGERMINATION MEDIA (BGM 2.5%) COMPOUND: QUANTITY PER LITER BT STOCK #1 10ml  BT STOCK #2 10 ml  BT STOCK #3 3 ml BT STOCK #4 3 ml BT STOCK #5 1ml SUCROSE 25 g   Adjust to pH 5.8. Dispense into sterile 1 L mediabottles ADDITIONS PRIOR TO USE: PER 1 L CEFOTAXIME (50 mg/ml) 2.5 mlFUNGICIDE STOCK    3 mlBT Stock for Bean Germination Medium

Make and store each stock individually. Dissolve each chemicalthoroughly in the order listed before adding the next. Adjust volume ofeach stock accordingly. Store at 4° C. Bt Stock 1 (1 liter) KNO₃ 50.5 gNH₄NO₃ 24.0 g MgSO₄*7H₂O 49.3 g KH₂PO₄ 2.7 g Bt Stock 2 (1 liter)CaCl₂*2H₂O 17.6 g Bt Stock 3 (1 liter) H₃BO₃ 0.62 g MnSO_(4—)H₂O 1.69 gZnSO₄—7H₂O 0.86 g KI 0.083 g NaMoO₄—2H₂O 0.072 g CuSO₄—5H₂O 0.25 ml of1.0 mg/ml stock CoCl₄—6H₂O 0.25 ml of 1.0 mg/ml stock Bt Stock 4 (1liter) Na₂EDTA 1.116 g FeSO₄7H₂O 0.834 g Bt Stock 5 (500 ml) Store in afoil wrapped container Thiamine-HCl 0.67 g Nicotinic Acid 0.25 gPyridoxine-HCl 0.41 g FUNGICIDE STOCK (100 ml) chlorothalonile (75% WP)1.0 g benomyl (50% WP) 1.0 g captan (50% WP) 1.0 g Add to 100 ml ofsterile distilled water. Shake well before using. BEAN ROOTING MEDIA(BRM) (for 4 L) MS Salts 8.6 g Myo-Inositol (Cell Culture Grade) 0.40 gSoybean Rooting Media Vitamin Stock 8 ml L-Cysteine (10 mg/mL) 40 mSucrose (Ultra Pure) 120 g pH 5.8 Washed Agar 32 g ADDITIONS AFTERAUTOCLAVING: BRM Hormone Stock 20.0 ml BRM HORMONE STOCK (1 liter) 6.0ml IAA (0.033 mg/ml) 4.0 ml SDW VITAMIN STOCK FOR SOYBEAN ROOTING MEDIA(1 liter) Glycine 1.0 g Nicotinic Acid 0.25 g Pyridoxine HCl 0.25 gThiamine HCl 0.05 g Dissolve one ingredient at a time, bring to volume.

The soybean plants are grown in a greenhouse to maturity, selfed andseeds collected. The seeds are planted in pots in conditions that permitgermination and seedling growth. The plants are treated with aneffective dose of the protoxin that corresponds to the conditionallethal gene. Leaf tissue samples are collected from the undamaged orsurviving seedlings and assayed for the reporter gene, for example, theGUS activity by MOPS method (see for example, Jefferson et al., EMBO J.6:3901, 1987). The seedlings are also assayed for the presence of the CP4 EPSPS enzyme by ELISA (Padgette et al. Crop Science 35:1451-1461,1995) or NPTII by ELISA depending on the construct used. The segregationratio is determined for the two T-DNAs. Plants with only the T-DNAcontaining the agronomic genes of interest are selfed, backcrossed oroutcrossed in accordance with standard breeding methods (Fehr,Principles of Cultivar Development Vol. 1, pp. 2-3 (1987).

DNA constructs containing two T-DNA molecules and at least oneconditional lethal gene and methods thereof are useful to enhance theidentification of transgenic plants for which the T-DNAs have integratedinto separate physical loci, and that can be genetically segregated ingametes and progeny. In addition, two DNA constructs each with at leastone T-DNA molecule and at least one of the DNA constructs having aconditional lethal gene, when present in different Agrobacterium strainsand transformed into plants by mixing the different strains, providesenhancement over co-transformation methods using strains of the sametype. Furthermore, the methods described in this example illustrate anaspect of the invention where when used in combination with the DNAconstructs of the invention are useful for the elimination of plantscontaining T-DNAs comprising marker transgenes, or the selectiveelimination of plants containing T-DNAs with agronomic transgenes or theselective elimination of germ cells containing T-DNAs with markertransgenes or agronomic transgenes. The method and DNA constructsenhance the frequency of detecting expression of the gene of interest,enhance the selection of independent segregation events and decrease thepresence of extraneous construct DNA segments in the plant genome.

Example 3

Strong constitutive promoters may influence the expression of linkedtransgenes that contain tissue or organ specific promoters, this resultsin an aberrant expression pattern from the tissue or organ specificpromoters referred to as leaky expression. The leaky expression of atransgene product in non-targeted tissues is an important issue fortransgenic plants, especially if the expression in the non-targettissues causes undesirable phenotypes. Additionally, often the trueexpression pattern and promoter strength of a tissue specific promotercannot be precisely determined because of the influence of a linkedexpression cassette containing a constitutive promoter. The presentinvention provides a method where DNA constructs that allow for theseparate integration of T-DNAs are used to reveal the true expressionpattern of tissue specific promoters. The method provides plants withtissue specific expression cassettes free from an expression cassettecontaining a selectable marker gene.

Seed tissue is an especially useful tissue for targeting a transgeneproduct. Pharmaceutical peptides, enzymes that enhance oil production orprovide for modified oils, enzymes that enhance vitamin production,enzymes that enhance essential amino acid content, enzymes that enhancestarch accumulation, proteins that provide more balanced amino acidcontent of the seed, enzymes that provide biopolymer production,proteins that are antimicrobial, antifungal, and insecticidal, alsomodified carbohydrate content, such as, low phytate, low stachyose, andenhanced fermentable sugars are some examples of products where seedspecific tissue expression maybe more desirable relative to constitutiveplant expression. Seed specific promoters, for example, the 7Sα′ andnapin promoters, from soybean and Brassica, respectively, can showactivity in vegetative tissues when the expression cassettes containingthese promoters and a gene of interest are linked to expressioncassettes containing constitutive promoters used to express selectablemarker genes. Promoters having constitutive activity are often isolatedfrom plant DNA viruses, e.g. caulimoviruses, such as a Figwort MosaicVirus (FMV) promoter or a cauliflower mosaic virus (CaMV) promoter aremost often used to direct expression of an antibiotic resistance geneproduct or herbicide resistance gene product. The NPTII antibioticenzyme (tolerance to kanamycin) and the EPSPS:CP4 enzyme (tolerance toglyphosate) are selectable marker gene products often used as fortransgenic plant production.

The DNA construct, pMON64200 (FIG. 19) is a 2 T-DNA vector that containsP-Gm.7Sα′/GUS/T-nos and P-FMV.35S/EPSPS:CP4/T-Ps.E9 expression cassettein separate T-DNAs that is transformed into soybean cells and fertileplants regenerated. The T-DNAs are integrated into the soybean genomeeither at different loci (unlinked) or at the same locus (linked). Inthis example, the gene of interest expresses β-glucuronidase (GUS) inseed tissue (cotyledon), however, the gene of interest could be any genefor which the expression product is desired to be expressed in a certaintissue or organ. The physical linkage between these two cassettes in R0and R1 plants is determined by using Southern blot and PCR analysis.Phenotypic segregation of the GUS activity and the glyphosate toleranceis also documented to confirm the physical linkage analysis. GUSactivity can be detected using a chromogenic substrate or the MOPS assaymethod and EPSPS:CP4 activity can be detected by spraying the plantswith glyphosate or using an immunological method to assay for thepresence of the EPSPS:CP4 protein. Because the R1 population is usuallysegregating, the GUS activity is examined in the cotyledons of thegerminating seeds to select plants that are positive for theP-Gm.7Sα′/GUS/F-nos cassette. Because the P-Gm.7Sα′ promoter is expectedto be seed specific, positive GUS activity in vegetative tissue, such asleaves, is used as the indicator of leaky expression.

Table 2 shows the analysis of transgenic events in which a two T-DNAconstruct is used to determine promoter expression patterns. A positive(+) for GUS staining in leaf indicates the plant has leaky expression, apositive (+) for GUS staining in cotyledon (speed specific expression)indicates the plant has a functional copy of P-Gm.7Sα′/GUS cassette, anda positive (+) glyphosate tolerance indicates that the plant has afunctional copy of the selectable marker cassette (P-FMV/EPSPS:CP4). Theevents are analyzed by Southern blot, phenotype, and PCR to determinethe linkage of the two expression cassettes. Three events have multipleinsertion loci that are identified as “locus 1 or locus 2” in thesegregating progeny. Ten events are shown to have linked expressioncassettes, 3 events are unlinked and 3 events are marker free. Table 2shows that all of the marker free or unlinked events do not show leafGUS expression, indicating that linkage with the selectable markerexpression cassette adversely affects the seed specific expressionpattern of the gene of interest. In contrast, 8 out of 10 linked eventsresults in a leaky expression pattern. TABLE 2 A summary of a 2T-DNAstudy on FMV/7Sα′ promoter interaction pMON64200 Leaf GUS Cotyledonglyphosate Linkage CP4/GUS Linkage event # (Leakiness) GUS toleranceSouthern phenotype PCR 20095-locus1 + + + NA linked linked 19931 + + +linked linked linked 19937 + + + linked linked linked 20139 + + + NAlinked linked 19927 + + + linked linked NA 19989 + + + NA linked linked20038 + + + linked linked linked 20043 + + + linked linked NA20286-locus1 − + + linked linked linked 20116 − + + linked linked NA20282 − + + unlinked segregating 20277 − + + unlinked segregating20127-locus2 − + − unlinked segregating 20095-locus2 − + − marker free20286-locus2 − + − marker free 20127-locus1 − + − marker freeNA—not available

For linkage PCR analysis, two primers of GUS coding region and twoprimers of EPSPS:CP4 coding region are designed to accommodate allpossible orientations. PCR reactions with four possible primercombination sets are performed for each linked event, and the likelyorientation is based on the primer set(s) that gave PCR products.

All of the unlinked and marker free plants show GUS expression only inthe cotyledon tissue indicating that linkage to the expression cassettecontaining the viral promoter negatively affects the tissue specificactivity of the Gm.7Sα′ promoter. Other tissue specific promoters knownin the art of plant molecular biology can be evaluated as described andused in the constructs and methods described herein to segregate thetransgene expression cassettes to provide enhanced function of thetissue specific promoter expression cassette.

Example 4

DNA constructs are created that contain four Ti plasmid border regionsthat comprise two T-DNA molecules, there is no substantial DNA segmentbetween the T-DNA molecules. The basic design of this plasmid is shownin FIG. 24. One of the T-DNA molecules contains the gene(s) of interest(GOI) and the second T-DNA molecule contains the plant selectable markergene and the plasmid maintenance elements. The Ti plasmid border regionsare arranged in like pairs with various configurations as illustrated inFIG. 25. The arrangement of border regions, GOI, plant positiveselectable marker gene, and plasmid maintenance elements in these DNAconstructs will provide for efficient independent integration of theT-DNA molecules.

Example 5

DNA constructs are created that contain two Ti plasmid border regions(RB, LB or combinations thereof) in which a gene of interest iscontained in a first DNA region between the two border regions that doesnot contain any plasmid maintenance elements, and in a second DNA regionbetween the two border regions contains a positive selectable markergene and plasmid maintenance elements (FIG. XX). The second DNA regioncontaining the plasmid maintenance elements is often referred to as thevector backbone. It has become undesirable to have vector backbonesequences in transgenic plants. However, in the constructs of thisaspect of the present invention the vector backbone becomes a DNA regionthat is integrated into the genome of a plant, this DNA region containsthe positive selectable marker gene and is intentional selected for inthe transgenic plants.

The constructs pMON65178 (FIG. 20), pMON65179 (FIG. 21), and pLagILB01(FIG. 23) illustrate the orientations of the plant expression cassettesin the DNA constructs with respect to the border regions and the plasmidmaintenance elements. The location of the positive selectable markergene is in a region of the plasmid that contains the plasmid maintenanceelements flanked by Ti plasmid border regions comprises a T-DNA, whereasthe gene of interest is located in a region of the plasmid flanked by Tiplasmid border regions that does not contain any functional plasmidmaintenance elements. These plasmids, and plasmids of similar design canbe transferred into plants by an Agrobacterium mediated method. Theresulting plants can be screened for the gene of interest and theselectable marker gene by methods that detect the presence of thetransferred DNA in the plant genome. These methods include: DNAamplification methods, e.g., polymerase chain reaction (PCR); and DNAhybridization methods, e.g., DNA Southern Blot analysis. The R₀Transgenic plants transformed with these constructs can be analyzed forthe linkage of the T-DNAs by digesting isolated plant genomic DNA withrestriction endonucleases that do not cut or cut rarely in any of theT-DNA segments, separating in an agarose gel by electrophoresis,transferring to a solid matrix and hybridizing with a labeledpolynucleotide probe creating a Southern blot that is exposed to a mediathat detects the labeled probe. The polynucleotide probe is selected tobe homologous to a DNA molecule specific to one of the T-DNAs. TheSouthern blot is then stripped of the first polynucleotide probe andhybridized with a second labeled probe that is specific to the otherT-DNA and exposed to a media that detects the labeled probe. Theresulting linkage Southern analysis can distinguish the linkagerelationship of the gene of interest (GOI) and the positive selectablemarker gene in the genome of the transgenic plant.

The constructs, pMON65178, pMON65179 and pMON65180 are transformed intocorn cells by an Agrobacterium mediated method and fertile plantsregenerated. In these constructs the GOI comprises a Zea mays Glb1promoter, a rice actin 1 intron, a Zea mays DHDPS transit signalpeptide, a dapA1 coding sequence (U.S. Pat. No. 6,329,574), and a Zeamays Glb1 transcriptional termination region. The selectable marker geneTable 3 illustrates the analysis of corn plants when analyzed by apolymerase chain reaction method for the GOI and linkage Southern blotanalysis of R0 plants and PCR analysis of the R1 population anddetermination of the percent of fertile plants that have progenysegregating for the GOI and the selectable marker gene. This analysisindicates that pMON65178 (RB/GOI/LB::EPSPS:CP4) and pMON65179(RB/GOI/RB::EPSPS:CP4) configurations are more effective than the morecomplex configuration of pMON65180 (RB/GOI/LB::RB/EPSPS:CP4/LB) forproviding plants with the GOI, 88.8%, 88.9% and 67.7%, respectively. Inaddition, the efficiency of the genetic element configurations in thepMON65178 and pMON65179 constructs and the method for providing usableplants with unlinked GOI and selectable marker gene is as good aspMON65180 (67.7%×17.2%=11.6%) relative to pMON65179 (88.9% X 12.4%=11%)and is better relative to pMON65178 (88.8%×33.7%=29.9%). TABLE 3Segregation analysis of 2 T-DNA containing corn plants UnlinkedSegregating event (R0 marker free pMON Configuration GOI Southern) R165178 RB/GOI/LB::CP4 88.8% 33.7% 100% (8/8) (119/134) (34/101) 65179RB/GOI/RB::CP4 88.9% 12.4%  86% (12/14) (232/261) (27/218) 65180RB/GOI/LB::RB/CP4/ 67.7% 17.2% LB (176/260) (21/122)

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim

1-55. (canceled)
 56. A DNA plasmid comprising a first Agrobacterium Tiplasmid right border region linked to at least one transgene ofagronomic interest linked to a second Agrobacterium Ti plasmid rightborder region linked to an antibiotic positive plant selectable markertransgene or a herbicide positive selectable marker transgene, linked toplasmid maintenance elements required for propagation in bacteria. 57.The DNA plasmid of claim 56, wherein said antibiotic positive plantselectable marker transgene or herbicide selectable marker transgene islinked to a conditional lethal transgene.
 58. The DNA plasmid of claim57, wherein said conditional lethal transgene is selected from the groupconsisting of: phosphonate monoester hydrolase, glyphosateoxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithinedeacetylase, P450 monooxygenase CPY105A1, and β-glucuronidase. 59-65.(canceled)
 66. The DNA plasmid of claim 56, wherein said antibioticpositive plant selectable marker transgene provides tolerance to anantibiotic is selected from the group consisting of hygromycin,kanamycin, bleomycin, G418, streptomycin and spectinomycin.
 67. The DNAplasmid of claim 56, wherein said herbicide selectable marker transgeneprovides tolerance to a herbicide selected from the group consisting ofglyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil,delapon, cyclohezanedione, a protoporphyrinogen oxidase inhibitor, andisoxaslutole herbicides.
 68. The DNA plasmid of claim 56, wherein saidtransgene of agronomic interest provides an agronomic trait comprisingherbicide tolerance, increased yield, insect control, fungal diseaseresistance, virus resistance, nematode resistance, bacterial diseaseresistance, mycoplasma disease resistance, modified oils production,high oil production, high protein production, germination and seedlinggrowth control, enhanced animal and human nutrition, low raffinose,environmental stress tolerance, increased digestibility, industrialenzyme production, pharmaceutical peptides and small moleculeproduction, improved processing traits, proteins improved flavor,nitrogen fixation, hybrid seed production, reduced allergenicity,biopolymers, or biofuel production.
 69. The DNA plasmid of claim 56,wherein said plasmid maintenance elements comprise at least one originof replication and an antibiotic marker.
 70. The DNA plasmid of claim69, wherein said origin of replication is a pBR322 origin of replicationand/or an oriV origin of replication from the RK2 plasmid.
 71. The DNAplasmid of claim 69, wherein said antibiotic marker is an aad (Spc/Str)gene for bacterial spectinomycin/streptomycin resistance.